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Colordao Bureau of Investigations SOP for DNA (June, 2002)

1. TPOX African Ca cssiah Southwest Southeast Allele American Hispanic Hispanic 209 oa N 209 N 240 gt 6 0 0120 0 0123 0 0120 0 0104 6 0 0861 0 0123 0 0120 0 0104 7 0 0215 0 0123 0 0120 0 0104 8 0 3684 0 5443 0 5550 0 5063 9 0 1818 0 1232 0 0335 0 0833 10 0 0933 0 0370 0 0335 0 0625 11 0 2249 0 2537 0 2727 0 2771 12 0 0239 0 0394 0 0933 0 0646 13 0 0120 0 0123 0 0120 0 0104 gt 13 0 0120 0 0123 0 0120 0 0104 THO1 African Ganesh Southwest Southeast Allele American Hispanic Hispanic N 210 N20 209 240 gt 5 0 0119 0 0123 0 0120 0 0104 5 0 0119 0 0123 0 0120 0 0104 6 0 1095 0 2266 0 2321 0 2125 7 0 4405 0 1724 0 3373 0 2521 8 0 1857 0 1256 0 0813 0 1042 8 3 0 0119 0 0123 0 0120 0 0104 9 0 1452 0 1650 0 1029 0 1854 9 3 0 1048 0 3054 0 2416 0 2354 10 0 0143 0 0123 0 0120 0 0104 Page 207 of 255 Appendix D Allele Frequencies CBI DNA Analysis SOP Vers 2 1 06 13 02 4 43 PM CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 ALLELE FREQUENCIES CONTINUED gt 10 0 0119 0 0123 0 0120 0 0104 D16S539 African Southwest Southeast Allele American caucasian Hispanic Hispanic mE N 209 N 202 N 208 N 240 gt 8 0 0120 0 0124 0 0120 0 0104 8 0 0359 0 0198 0 0168 0 0229 9 0 1986 0 1040 0 0793 0 1458 10 0 1101 0 0668 0 1731 0 0958 11 0 2943
2. Date Prepared Amount of tubes 30 H20 lot Preparer 30 H202 Page 238 of 255 Appendix AE DNA Working Solutions Log 30 Hydrogen Peroxide CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 APPENDIX AF DNA WORKING SOLUTIONS LOG ORGANIC SPERM WASH BUFFER MODEL 10MM TRIS HCL 10MM EDTA 50 MM NACL 2 SDS PH 8 0 500 Solution Preparation Add 5 1M Tris HCl 8 0 10 0 5M EDTA pH 8 0 0 05 5 ml 5M NaCl or use 0 29 NaCl and 50 20 SDS to 430 ml ultrapure water adjust volume to 500 Check pH Autoclave Store at room temperature Date 1M Tris HCl pH 0 5M EDTA 5M NaCl 20 SDS Amount Prepared 8 0 lot lot loti loti pH Preparer Verified date ORGANIC SPERM WASH BUFFER Page 239 of 255 Appendix AF DNA Working Solutions Log Organic Sperm Wash Buffer Model CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 APPENDIX AG DNA WORKING SOLUTIONS LOG ORGANIC STAIN EXTRACTION BUFFER MODEL 10MM TRIS 100MM NACL 10MM EDTA 2 SDS 1L Solution Preparation Dissolve 5 84 g NaCl in 500 ml ultrapure water by stirring To this solu
3. 201 ee eA AA Ee Ed 202 ue M LL e M LM 203 e tas 204 DY Gots CECE REY Rta E rne La MEE QM LM Pre 205 LI E S UR 206 IROU ecc 207 iSo EET ERIT 207 VAG 208 WORKSHEETS cc hich thre 209 DNA QUANTITATION 210 DNA SUMMARY MODEL eterne tentent nti 211 AMPLIFICATION DATA 212 FREQUENCIES 213 STR DILUTION AND AMPLIFICATION WORKSHEET MODEL 214 GENESCAN WORKSHEET MODEL eet 215 MIXTURE ANALYSIS WORKSHEET PROFILER PLUS MODEL 216 MIXTURE ANALYSIS WORKSHEET COFILER MODEL 218 DNA STOCK SOLUTIONS LOG 8 uG uL BOVINE SERUM ALBUMIN BSA 219 DNA STOCK SOLUTIONS LOG 10X CITRATE BUFFER MODEL 220 DNA STOCK SOLUTIONS LOG 0 5M EDTA PH 8 0 MODEL 221 DNA STOCK SOLUTIONS LOG 5M NACL MODEL 223 DNA STOCK SOLUTIONS LOG PBS BUFFER PH 7 4 PHOSPHATE BUFFER SALINE 220040 4 224 DNA STOCK SOLUTIONS LOG 1M SODIUM ACETATE 5 2 MODE nina E Mud M E 225 DNA STOCK SOLUTIONS LOG 20 W V SODIUM DODECYL SULFATE 2 226 DNA STOCK SOLUTIONS LOG 20X SSPE BUFFER MODEL 227
4. N 180 N 199 N 203 N 240 lt 7 0 0139 0 0128 0 0123 0 0104 7 0 0139 0 0128 0 0616 0 0229 8 0 0500 0 0128 0 0123 0 0104 9 0 0139 0 0308 0 0542 0 0500 10 0 0639 0 0487 0 0665 0 0458 11 0 2611 0 4103 0 4212 0 3938 12 0 3556 0 3539 0 2906 0 3167 13 0 2444 0 1462 0 0961 0 1542 14 0 0139 0 0128 0 0123 0 0104 15 0 0139 0 0128 0 0123 0 0104 gt 15 0 0139 0 0128 0 0123 0 0104 Page 203 of 255 Appendix D Allele Frequencies CBI DNA Analysis SOP Vers 2 1 06 13 02 4 43 PM CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 ALLELE FREQUENCIES CONTINUED 0138317 African Southwest Southeast Allele American mE N 179 N 198 N 203 N 240 lt 8 0 0140 0 0128 0 0123 0 0104 8 0 0363 0 0995 0 0665 0 1146 9 0 0279 0 0765 0 2192 0 1146 10 0 0503 0 0510 0 1010 0 0771 11 0 2374 0 3189 0 2020 0 3063 12 0 4832 0 3087 0 2168 0 2292 19 0 1257 0 1097 0 1379 0 1083 14 0 0363 0 0357 0 0567 0 0500 15 0 0140 0 0128 0 0123 0 0104 gt 15 0 0140 0 0128 0 0123 0 0104 Page 204 of 255 Appendix D Allele Frequencies CBI DNA Analysis SOP Vers 2 1 06 13 02 4 43 PM CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 ALLELE FREQUENCIES CONTINUED D7S820 African Southwest Southeast Allele American caucasian Hispanic Hispanic mE N 210 N 203 N 20
5. R 9947A Identifiler 0851179 123 170 8 19 pa 13 13 24 24 2 25 28 28 2 29 29 2 30 D21S11 185 240 6 FAM 30 30 30 2 31 31 2 32 32 2 33 3 Blue 3 2 34 34 2 35 35 2 36 38 078820 255 292 6 15 444 Blue CSF1PO 305 342 6 15 6 042 Blue VIC D3S1358 112 140 12 19 14 15 Green VIC THO1 163 202 4 9 9 3 10 11 13 3 8 9 3 t Ea Green VIC 0138317 217 245 8 15 RE 11 11 0165539 252 293 5 8 15 VIC 11 12 Green VIC 0251338 307 359 15 28 19 23 Green g2149213 13914 NED 0198433 102 135 14 15 14 2 15 15 2 16 16 2 17 1 Yellow 7 2 Page 126 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 Table 6 SIZE DYE POSITIVE SYSTEM RANGE SES IN COLO CONTROL bp R 9947A NED Identifiler 155 207 11 24 Yellow 17 18 TPOX 222 250 6 13 NED Yellow 7 9 10 10 2 11 13 NED 018551 262 345 13 2 14 14 2 15 27 Yellow PET Red PET Red 8 8 Amelogenin 107 113 X Y 055818 134 172 7 16 17 26 26 2 27 30 30 2 215 355 31 2 32 2 33 2 42 2 43 2 44 2 45 2 46 2 Red 47 2 48 2 50 2 51 2 Sizes are actual base pair size of alleles the Identifiler ladder The sizes obtained in Genotyper may vary from the actual fragment sizes due to electrophoretic effects The allelic ladders provided in the kit are used to determine the genotypes of the sample The alle
6. 0 04 Bromo Blue lot Preparer QBSPOT Page 244 of 255 Appendix AK DNA Working Solutions Log QuantiBlot Spotting Solution CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 APPENDIX AL DNA WORKING SOLUTIONS LOG QUANTIBLOT WASH SOLUTION MODEL 1 5X SSPE 0 5 wiv SDS 2L Solution Preparation Add 150 20X SSPE 50 20 w v SDS to 1800 ml ultrapure water and mix thoroughly Note Wash solution solids must be in solution before use warming e g 50 C incubator may be required to dissolve solids completely Preparation in a clear glass container is recommended to facilitate visual inspection for solids during warming 20X SSPE 20 SDS Date Prepared Amount lot lot Preparer QBWASH Page 245 of 255 Appendix AL DNA Working Solutions Log QuantiBlot Wash Solution CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 APPENDIX AM DNA WORKING SOLUTIONS LOG QUANTIBLOT PM DQA1 HYBRIDIZATION SOLUTION MODEL 5X SSPE 0 5 w v SDS 1L Solution Preparation Add 250 20X SSPE and 25 20 SDS w v to 725 u
7. 10 ML Solution Preparation Add 4 0 ml 0 5M EDTA to 6 0 ml ultrapure water and mix thoroughly The solution may be divided into 300 ul aliquots using microcentrifuge tubes Date Prepared Amount 0 5M EDTA lot Preparer 200MEDTA Page 236 of 255 Appendix AC DNA Working Solutions Log 200mM EDTA CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 APPENDIX AD DNA WORKING SOLUTIONS Loc 3 HYDROGEN PEROXIDE MODEL Solution Preparation Add 1ml 30 H2O to 9ml sterile ultrapure water and vortex to mix The solution may be divided into 400 ul aliquots using microcentrifuge tubes Protect from light Store at 2 to 8 C Note 3 Hydrogen Peroxide expires 1 month after preparation Date Prepared Amount 30 H20 lot Preparer 3 H202 Page 237 of 255 Appendix AD DNA Working Solutions Log 3 Hydrogen Peroxide Model CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 APPENDIX AE DNA WORKING SOLUTIONS LOG 30 HYDROGEN PEROXIDE MODEL Solution Preparation Aliquot 5 ml 30 H2O into 15 ml tubes and store at 4 C in area where QuantiBlot is performed
8. Samples displaying split peak characteristics indicates excess target DNA may be incubated again at 60 C to complete the A addition diluted and then re typed NOTE Microvariants may differ by only one base pair so interpretation should be made with caution Microvariants will not change base pair value upon re amplification and retyping NOTE Shoulder peaks occur at the Amelogenin locus in the n position here n 1 Amelogenin These shoulders are not due to insufficient nucleotide addition of the PCR product but rather a phenomenon of capillary electrophoresis These peaks do not interfere with interpretation at this locus Page 113 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 PULL UP Small artifactual peaks can appear in other colors under true peaks This phenomenon is termed pull up Pull up is the result of spectral overlap between the dyes which are normally corrected for by the Matrix If a pull up peak is above the minimum peak height detection threshold it will be sized at approximately the same size as the true peak Pull up can occur as a result of the following A application of a sub optimal matrix and B amplification using excess input DNA which may lead to off scale peaks SPIKES AND OTHER ANOMALIES Transient fluorescent materials in the injection i e precipitated urea fabric dyes air bubbles etc as well as electrical impulses can
9. 69 69 PREPARATION OF PCR PRODUCT FOR 72 DNA HYBRIDIZATION 4 4 4 tente re eR e ERE ena 72 COLOR DEVELOPMENT gt eoe 76 PHOTOGRAPHYSAND STORAGE tenu tat tette 78 DISPOSAL OR REUSE OF TYPING 78 INTERPRETATION OF RESULTS 2 78 PERFORMANCE CHARACTERISTICS 82 TROUBLESHOOTING c cene e eot e 82 ALLELE FREQUENCIES DQAT PM iata et eia a aia ruere ae 92 SHORT TANDEM REPEAT ANALYSIS ei nite adiens eee 94 PRINCIPLES OF STR ANALYSIS 94 GUIDELINES FOR CONTROL SAMPLES ono iiti iiec rtis cune tates 96 EXTRACTION CONTROLS 4a setate teda n e DR 96 AMPLIFICATION CONTROLS ioter tete SE oA 97 Page 3 of 255 Table of Contents Continued CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 II 97 QUALITY CONTROL OF CRITICAL SUPPLIES AND REAGENTS 9
10. APPENDIX AQ DNA CHEMICAL Loc MODEL Chemical Reagent Supplier Catalog Lot Received Expiration Date Page 250 of 255 Appendix AQ DNA Chemical Log Model CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 APPENDIX AR DNA QA QC Form MODEL Commercial Kit Purchased From DATE DATE RECEIVED OPENED VERIFIED BY DATE DNA QAQC FORM Page 251 of 255 Appendix AR DNA QA QC Form Model CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 APPENDIX AS EXTRACTION SUMMARY SHEET MODEL Analyst Date Concentrate Case Item Description Volume az Comments Reagent Lot 1 2 3 4 Proteinase K 5 20 Chelex 6 Digest Buffer 7 8 Tris EDTA NaCl 9 20 Sarkosyl 10 Org Sp Wash 11 12 13 14 15 16 17 18 19 20 CEC Cut Extract Control E Epithelial Fraction S Sperm fraction T Tissue Org Sp Wash Organic Sperm Wash EXTRACTION SUMMARY SHEET Page 252 of 255 Appendix AS Extraction Summary Sheet Model CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 Identifiler kit lot JUNE 2002 APPENDIX AT CBI DNA DATABASE SAMPL
11. Add QuantiBlot 01721 Probe Add Enzyme Conjugate HRP SA at indicated step in protocol Use 180 tl Enzyme Conjugate HRP SA for colorimetric TMB detection Prepare new Color Development Solution using a fresh bottle of hydrogen peroxide Concentrations of MgCl gt 0 3 result reduced sensitivity Pre pare all DNA dilutions in TE Buffer Any MgCl be removed from samples by microdialysis using Centricon 100 spin units follow manufacturer s di rections Reduce the rotation rate of the water bath to 50 60 rpm Check that the membrane is fully sub merged in the bottom of the Hybridization Tray before shaking Do not allow the mem brane to dry at any point in the protocol CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 OBSERVATION 3 Non uniform signal intens ity within a slot 4 Filter background JUNE 2002 POSSIBLE CAUSE Bubble s in slot blot wells when sample was pipetted into well or when vacuum was applied No or low SDS in the Hybridization Solution or in the QuantiBlot Wash Solu tion Membrane was not pre wetted prior to slot blotting Too much Enzyme Conju gate HRP SA was added Page 64 of 255 CBI DNA Analysis SOP Vers 2 1 RECOMMENDED ACTION Slowly pipet the Spotting Solution directly over the center of the wells of the slot blot apparatus with the pipette tip raised approximately 5mm above the membrane Turn on t
12. rocking the tray for several seconds then aspirate the solution from each well It is important to remove any solution from the tray lid with a clean lab wipe at this point to prevent excess blue background coloration from forming during the color development procedure 12 perform the stringent wash step dispense 5 ml pre warmed PM DQA1 Wash Solution into each well Cover the tray with the clear plastic lid and place it in the 55 C water bath Place a 1 kg weight on the covered tray to prevent the tray from sliding or floating Adjust the rotation to 50 to 90 rpm and check the tray position to insure that water does not splash into the wells of the tray U Replace the water bath cover to maintain the temperature at 55 C 1 C Incubate the DNA probe strips at 55 C 1 C for 12 minutes 1 minute The temperature and timing of the stringent wash step are critical V After incubation remove the tray from the water bath Take off the lid and aspirate the contents of each well from the labeled end of the strips Remove any liquid from the tray lid with a clean lab wipe W Dispense 5 ml of PM DQA1 Wash Solution into each well Cover and rock the tray gently for several seconds X Remove the lid and slowly pour off or aspirate the contents from each well into a designated waste container Remove any solution from the tray lid with a clean lab wipe COLOR DEVELOPMENT A Dispense 5 ml Citrate Buffer into each well
13. A B C D F Add 20 ul TE Buffer to each database sample tube Dispense 30 ul of Master Mix into each reaction tube including those for the positive control reagent blank and negative amplification blank If using the 480 Thermal Cycler add one drop of mineral oil Supplied with kit to each of the tubes Add 20 ul of the positive control to the positive control tube add 20 ul of TE Buffer to the reagent blank and negative amplification blank tubes If using the 9700 Thermal Cycler you may centrifuge the tubes briefly The samples are now ready for amplification SAMPLE PREPARATION FTA WASHED SAMPLE FOR FTA AND S amp S PAPER SAMPLES USING IDENTIFILER KIT A B 10 ul TE Buffer to each database sample tube Dispense 15 ul of Master Mix into each reaction tube including those for the positive control reagent blank and negative amplification blank Add 10 of the positive control to the positive control tube for a 28 cycle PCR add 10 ul of TE Buffer to the reagent blank and negative amplification blank tubes When using NIST traceable CBI internal control as the positive control add 10 ul TE to the tube If using the 9700 Thermal Cycler centrifuge the tubes briefly if necessary The samples are now ready for amplification Page 133 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 AMPLIFICATION A Place the PCR tubes in the
14. Dealing with Contamination Enzymatic Control of Carryover Contamination in PCR PCR Meth amp Appl Suppl 1992 510 514 Innis Michael A et al eds PCR Protocols A Guide to Methods and Applications New York Academic Press 1990 Kirby Lorne T ed DNA Fingerprinting An Introduction New York Stockton Press 1990 Kunkel T DNA Replication Fidelity Jour Biol Chem 267 1992 18251 4 Krawckak Reiss J Schmidtke J Rosler U Polymerase Chain Reaction Replication Errors and Reliability of Gene Diagnosis Nuc Acids Res 17 1989 2197 201 Kwok S Higuchi R Avoiding False Positives with PCR Nature 339 1989 237 8 Saliki Randall K et al Enzymatic Amplification of B globin Sequences and Restriction Site Analysis for Diagnosis of Sickle Cell Anemia Science 230 1985 1350 4 Tindall R Kunkel A Fidelity of DNA Synthesis by the Thermus aquaticus DNA Polymerase Biochemistry 27 1988 6008 13 Walsh P S Erlich G A Higuchi R Preferential PCR Amplification of Alleles Mechanisms and Solutions PCR Meth amp Applic 1 1992 241 50 STR ANALYSIS ABI PRISM 310 Genetic Analyzer User s Manual Rev 1 1995 ABI PRISM 310 Genetic Analyzer User s Manual 1998 ABI PRISM 377 DNA Sequencer User Manual 1998 Page 191 of 255 Appendix C Resources CBI DNA Analysis SOP Vers 2 1 06 13 02 4 43 PM CBI FORENSIC LABORATORY DNA ANALY
15. Determines whether or not two DNA profiles match CODIS supports three levels of match stringency low moderate and high Low Matches occur when or more allele match between the target and candidate profiles at a given locus All alleles do not have to match at low stringency Moderate Matches require all alleles to match but the target and candidate profiles can contain a different number of alleles That is if the target profile has two then two alleles must match If the target profile has just one allele then a candidate may have two alleles This match stringency is used to search the Forensic Mixture Database High All alleles must match between the target and candidate profiles This match stringency is used to search the Forensic Unknown Database NDIS National DNA Index System The FBI administered centralized system of DNA records contributed by all State and Local participating laboratories NDIS receives records from every lower level index and supports the searching functions at a National level NDIS Audit Review Board A board convened by the FBI that has the responsibility of reviewing external audit results from NDIS participating laboratories Non Suspect Case A case is considered a non suspect case when it falls into one of the following categories 1 A case without a suspect listed that is analyzed for DNA 2 A case in which DNA analysis has been conducted and the listed suspect is
16. FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 APPENDIX J GENESCAN WORKSHEET MODEL CASE FILE ANALYST IDENTIFIER SIZE DATY COMMENTS Delete HEIGHT POINT OLA Off Ladder Allele PU Pull Up B Blue G Green Y Yellow R Red DP Dye Peaks M manual delete A computer delete D deleted run from Genotyper A incomplete A addition RB Reagent Blank RBS Reagent Blank Sperm RBE Reagent Blank Epithelial NAB Negative Amplification CECT Cut Extraction Control Tissue CECS Cut Extraction Control Sperm CECE Cut Extraction Control Epithelial PC Positive amplification Control Page 215 of 255 Appendix J GeneScan Worksheet CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 APPENDIX K MIXTURE ANALYSIS WORKSHEET PROFILER PLUS MODEL Case Item Date Peak Height Interpretation and Comments rise T observed TE um A major FGA Page 216 of 255 Appendix K Mixture Analysis Worksheet Profiler Plus Model CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 Page 217 of 255 Appendix K Mixture Analysis Worksheet Profiler Plus Model CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 APPENDIX L MIXTURE ANALYSIS WORKSHEET COFILER MODEL Case Item Analyst
17. Hispanic Hispanic m 210 196 N 191 N 191 lt 12 0 0119 0 0123 0 0120 0 0131 12 0 0119 0 0123 0 0120 0 0131 13 0 0119 0 0123 0 0120 0 0131 14 0 1214 0 1404 0 0790 0 0838 15 0 2905 0 2463 0 4258 0 3534 15 2 0 0119 0 0123 0 0120 0 0131 16 0 3071 0 2315 0 2656 0 2461 17 0 2000 0 2118 0 1268 0 1623 18 0 0548 0 1626 0 0837 0 1387 19 0 0119 0 0123 0 0144 0 0131 gt 19 0 0119 0 0123 0 0120 0 0131 2 These frequency tables are from the Short Tandem Repeat Analysis Protocol Federal Bureau of Investigation Laboratory 7 00 Page 197 of 255 Appendix D Allele Frequencies CBI DNA SOP Vers 2 1 06 13 02 4 43 PM CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 ALLELE FREQUENCIES CONTINUED VWA African Southwest Southeast Allele American 180 2196 203 240 lt 11 0 0139 0 0128 0 0123 0 0104 11 0 0139 0 0128 0 0123 0 0104 12 0 0139 0 0128 0 0123 0 0104 13 0 0139 1 0128 0 0123 0 0104 14 0 0667 0 1020 0 0616 0 0688 15 0 2361 0 1122 0 0764 0 1000 16 0 2694 0 2015 0 3596 0 2688 17 0 1833 0 2628 0 2217 0 3042 18 0 1361 0 2219 0 1946 0 1875 19 0 0722 0 0842 0 0714 0 0500 20 0 0278 0 0128 0 0123 0 0167 21 0 0139 0 0128 0 0123 0 0104 gt 21 0 0139 0 0128 0 0123 0 0104 198 of 255 Appendix Allele Frequencies CBI DNA Analysis SOP V
18. Profiler Plus Profiler Plus or PP AmpF STRO Cofiler Cofiler or and AmpF STR Identifiler or ID DNA typing systems Applied Biosystems utilize the polymerase chain reaction PCR to amplify regions of DNA known as short tandem repeats STRs in order to characterize DNA extracted from database specimens Profiler Plus Cofiler and Identifiler are multiplex systems which allow for the simultaneous amplification of numerous STR loci as well as a portion of the amelogenin gene located within the X and Y chromosomes Analysis of amelogenin allows for gender determination The Profiler Plus Cofiler and Identifiler kits contain all reagents needed for amplification which includes primer sets which are specific for the various loci PCR reaction buffer and AmpliTaq Gold DNA polymerase as well as the required allelic ladders The primer sets contained within each kit consist of both unlabeled primers and those that are labeled with one of three PP CO or four ID distinctive fluorescent dyes The use of multicolor dyes permits the analysis of loci with overlapping size ranges as well as the inclusion of a sizing standard with each sample The amplified fragments are separated according to size by electrophoresis using the ABI Prism 377 DNA Sequencer or ABI Prism 3100 Genetic Analyzer The fluorescent dyes labeling the amplified fragments are detected by laser excitation and a charge coupled device CCD camera The re
19. REAGENTS DNA analysis reagents are reagent grade or better A log is maintained which records and inventories reagents date chemicals reagents received supplier catalog number lot number storage location and expiration date where appropriate The in house reagents are labeled as to content and concentration lot date of preparation and initials of preparing analyst and expiration date expiration date must be present if no expiration date is required then write No expiration date A log is maintained for in house reagents which records date of preparation the amount made lot numbers of chemicals used the preparer and an expiration date Formulation procedures for reagents standards and controls are also included at the top of each reagent log form and in the CBI Forensic Laboratory DNA SOP Manual A log is maintained of commercial amplification kits quantitation kits CRITICAL REAGENTS The DNA Laboratory identifies critical reagents and verifies them prior to use in casework and database analyses These critical reagents include commercial kits for performing genetic typing for casework analysis reagents used extraction of casework samples e FTA paper for database Amplification reagents are commercially prepared and supplied with the Amplitype AmpF STR Profiler Plus AmpF STR Cofiler kits and AmpF STR Identifiler kits They are quality controlled by the manufacturer prior to release
20. The case file s for the cases involved will be obtained The analysts involved with the cases will be notified The analyst s who processed the case will review the DNA profile s entered into CODIS for confirmation If the hit involves a convicted offender profile the sample from that individual will be re extracted and re analyzed to confirm the match Investigating officers will be notified The case reports will reflect that the officers were notified of the hit A copy of the CODIS match report will be placed into each of the case files The hit will be recorded on the CODIS hits worksheet as well as the hit scorecard The State CODIS Administrator in Denver will be notified by e mail of any hits made at the Local level The hit will be added to the State hit total Copies of the following will be forwarded to the State CODIS Administrator the CODIS match report the case report the STR allele worksheet the case submittal sheet and any written correspondence regarding the hit Page 171 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 If a possible hit occurs at the State level SDIS 1 The State Administrator will inform the Local Administrator A copy of the hit report will be forwarded or a match message will be sent The DNA analyst s involved will be notified so that the DNA profiles can be reviewed If the match involves a convicted offender profile
21. Tubes not seated tightly in the DNA Thermal Cycler 480 block during amplification Hybridization or Stringent Wash temperature too high Hybridization or PM DQA1 Wash Solution salt concentration too low Stringent Wash time too long Amplified DNA was not added to DNA probe strips Amplified DNA was not denatured Enzyme Conjugate HRP SA was not added to the Enzyme Conjugate Solu tion Page 83 of 255 CBI DNA Analysis SOP Vers 2 1 DNA ANALYSIS SOP VERSION 2 1 RECOMMENDED ACTION See GeneAmp PCR Instrument System Manual and check instrument calibration Push tubes firmly into contact with block after first cycle repeat test Check that the rotating water bath temperature is 55 C 1 C repeat test Prepare new solutions repeat test Repeat test washing for 12 minutes 1 minute only Repeat test adding amplified DNA to DNA probe strips Check GeneAmp PCR Instrument System block temperature 95 C and leave sample in block gt 3 minutes complete DNA addition to tray within 20 seconds for each sample repeat test Prepare new diluted Enzyme SA solution repeat test CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 OBSERVATION 2 Positive signal from Control DNA 1 but no signal from DNA test sample 3 High DNA probe strip background color 4 High DNA probe strip background color upon storage JUNE 2002 POSSIBLE
22. A dispensing re pipette is useful for this purpose Cover the tray with the clear plastic lid and place it on an orbital shaker set at approximately 50 rpm at room temperature 15 to 30 C for 5 minutes Page 76 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 B During this wash step prepare the Color Development Solution Do not prepare the Color Development Solution more than 10 minutes before use Add the following reagents in the order listed to a glass flask and mix thoroughly by swirling Protect from light Do NOT vortex Use the following equations to determine the volumes of each component required Number of 5ml Volume of Strips X Citrate Buffer Citrate Buffer Number of 5 ul Volume of Strips X 396 Hydrogen Peroxide Hydrogen Peroxide Number of 0 25 ml Volume of Strips X Chromogen TMB Solution Chromogen TMB C Remove the tray from the orbital shaker Remove the lid and slowly pour off or aspirate the contents from each well into a designated waste container Add 5 ml freshly prepared Color Development Solution Step B to each well D Develop the strips at room temperature 15 to 30 C by rotating on an orbital shaker set at approximately 50 rom for approximately 20 to 30 minutes NOTE Place the clear plastic lid on the tray and cover the lid with aluminum foil during Steps D F and G to protect the DNA probe strips from strong light
23. DNA STOCK SOLUTIONS LOG 10X TBE 4 4 00 2 228 DNA STOCK SOLUTIONS LOG 1M TRIS HCL PH 7 5 MODEL 229 Page 8 of 255 Table of Contents Continued CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 DNA STOCK SOLUTIONS LOG 1M TRIS HCL 8 0 MODEL 230 DNA WORKING SOLUTIONS LOG 5 W V CHELEX SOLUTION MODEL e 231 DNA WORKING SOLUTIONS LOG 20 W V CHELEX SOLUTION MODEL 232 DNA WORKING SOLUTIONS LOG DEIONIZED FORMAMIDE MODE if cinerea en Ru D uL AC es 233 DNA WORKING SOLUTIONS LOG DIGEST BUFFER MODEL 234 DNA WORKING SOLUTIONS LOG 1M DTT DITHIOTHREITOL 10MM SODIUM ACETATE PH 5 2 MODEL 235 DNA WORKING SOLUTIONS LOG 200MM EDTA 236 DNA WORKING SOLUTIONS LOG 3 HYDROGEN PEROXIDE ipi ed teint 237 DNA WORKING SOLUTIONS LOG 30 HYDROGEN PEROXIDE MODEL Dub UM 238 DNA WORKING SOLUTIONS LOG ORGANIC SPERM WASH cr 239 DNA WORKING SOLUTIONS LOG ORGANIC STAIN EXTRACTION BUFFER MODEL ctr i d eames 240 DNA WORKING SOLUTIONS LOG PM DQA1 W
24. E Remove the tray from the shaker Remove the lid and slowly pour off or aspirate the contents from each well into a designated waste container F Stop the color development by washing the strips in deionized or glass distilled water DI HzO Dispense approximately 5 ml DI into each well Place the tray an orbital shaker set at approximately 50 for 5 to 10 minutes Repeat Section E G Repeat Section F at least two times for a minimum of three DI H2O washes Additional 5 to 10 minute washes will reduce the potential for development of background color H Record the pattern of blue dots from each wet AmpliType PM DNA Probe Strip see Interpretation of Results NOTE Keep strips wet throughout the photography steps Page 77 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 PHOTOGRAPHY AND STORAGE A Photographs shall be taken for a permanent record of the results Photographs must be taken while DNA probe strips are still wet B Place wet strips on a flat non absorbent surface a black background is recommended to enhance contrast Keep the strips wet throughout the photography steps Minimize exposure to strong light C Use a Polaroid camera with Polapan 400 or Type 52 black and white film or Type 59 or 559 color film or other suitable recording device NOTE For black and white photography an orange filter Wratten 22 or 23A will enhance con
25. EDTA must be added prior to heat denaturation of the samples Samples are now ready for DNA hybridization and color development Amplified samples containing 9 5mM EDTA may be stored at 2 to 8 for 2 months or at 20 C for 6 months The continued acceptable performance of these samples beyond these periods may vary with the sample Store amplified DNA samples separate from all PCR amplification reagents extracted DNA samples and casework samples DNA HYBRIDIZATION The AmpliType PM DQA1 DNA Hybridization process involves 3 steps performed sequentially as follows 1 hybridization of amplified DNA to DNA probe strips 2 binding of HRP SA to hybridized PCR products and 3 stringent wash to remove non specifically bound PCR products Color Development follows the stringent wash step Certain steps in the following procedures involve the aspiration of solutions con taining amplified DNA Before starting the DNA Hybridization and Color Development procedures assemble the required reagents and equipment as follows Enzyme Conjugate HRP SA included in kit Chromogen TMB Solution included in kit prepared in Section A Hybridization Solution PM DQA1 Wash Solution Citrate Buffer 3 Hydrogen Peroxide AmpliType PM DQA1 DNA Probe Strips Included in Kit AmpliType DNA Typing Trays Part N808 0065 Page 72 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE
26. OFF LADDER ALLELES 154 PEAK HEIGHT 4 44 0000 155 TRAL ELC P ROR E S oe e RR e euet em MU E 155 SPILLOVER AND LEAKAGE ABI cute CE Duce DUC DUCES 156 REAGENT BLANKS nnna Nuus 156 NEGATIVE AMPLIFICATION BLANKS 7 156 FOSIE CONT ROS saree d REN IR E E t A a ak a te 156 MERGING GENOTYPER ALLELIC TABLE FILES AFTER TECHNIGAL REVIEW 156 CREATING GMF FILES 5 lee dee 157 CREATING CMF FILE USING NT 5 218121 157 CREATING A CMF FILE USING MACINTOSH 158 ANALYST AGTIVITM uicit ee aptid ubi aid p dopo tet prb od mites dea 159 NAMING CONVENTION FOR STR ANALYSIS OF DATABASE OAMP chofer 160 MACINTOSH FILE NAMES odes de tbe e bit D ERO pdt eu D ola 160 WINDOWS NT FILE NAMES i ri eot etn rd ue e CE E e ere ne 160 RTT 160 REFERBENGES Goa et a E a a em av td du ah d 161 CODIS oeste oc note tod p pte pto a a t d Dd Sd duque 163 INTRODUCTION TO GODS s 5 Ebo Eb e 163 ORGANIZATION AND MANAGEMENT 164 FORENSIC DNA ANALY Kod fond fone fan fant Jan aie an te E 164 DA
27. PM DQA1 WASH SOLUTION 2 5X SSPE 0 1 w v SDS 2L Add 250 ml 20X SSPE and 10 ml 2096 w v SDS to 1 740 ml ultrapure water and mix thoroughly Store at room temperature Wash solution solids must be in solution and the solution must be well mixed before use warm in an incubator to 50 55 C to dissolve solids completely and prior to use Preparation in a glass container is used to facilitate visual inspection for solids during warming PRE WETTING SOLUTION 0 4N NaOH 25mM EDTA 500 ml Add 20 ml 10N NaOH 25 ml 0 5M EDTA to 455 ml ultrapure water and mix thoroughly Store at room temperature 10 mg ml PROTEINASE K 10 ml Dissolve 100 mg Proteinase K in 10 ml sterile ultrapure water Aliquot solution 0 5 ml and store 0 5 ml aliquots frozen at 20 C QUANTIBLOT WASH SOLUTION 1 5X SSPE 0 5 w v SDS 2L Add 150 20X SSPE and 50 20 w v SDS to 1 800 ml ultrapure water and mix thoroughly Wash solution solids must be in solution before use warm to 50 55 C in an incubator to dissolve solids completely prior to use Prepare in a glass container to facilitate visual inspection for solids during warming Page 29 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 20 SARKOSYL N Laurylsarcosine 100 ml Add 20 N Laurylsarcosine to ultrapure water and stir until dissolved Bring to a final volume of 100 ml with sterile ultrapure water
28. cutting to tube Incubate at 4 C for 2 hours or overnight Twirl the swab with a sterile toothpick for at least 2 minutes to agitate the cells off of the substrate Remove the swab and toothpick Store swab or fabric in a sterile tube A Costar spin basket may be utilized for the agitation of the cellular component from the substrate See page 13 E OPTIONAL Spin in a microcentrifuge for 2 minutes at 10 000 to 15 000 x g Without disturbing the pellet remove all but 50 ul of the supernatant using microtip disposable transfer pipette and place into the tube containing the swab or fabric This supernatant can be used for traditional serological analyses if necessary Re suspend the pellet in the remaining 50 ul by stirring with a sterile pipette tip OPTIONAL remove about 3 ul of the re suspended sample and spot on glass microscope slide for examination Perform Christmas Tree Stain Serology SOP Staining of Spermatozoa Kernechtrot Picroindigocarmine NOTE If sperm heads are detected follow the spermatozoa extraction in the presence of contaminating cells protocol beginning with Step If sperm are not detected the sample may not be useable BUCCAL SCRAPINGS A Scrape the inside of the cheek with a toothpick B Twirl the toothpick directly into 200 ul of 5 Chelex in a sterile 1 5 ml microcentrifuge tube C Follow protocol for Inorganic Extraction Chelex Blood Procedure beginning with Step F BU
29. disturb the cell pellet H Repeat Step G two additional times for a total of three washes of the cell pellet NOTE Additional wash steps are recommended when the ratio of sperm to epithelial cells is low l Re suspend the pellet by stirring with a sterile pipette tip Remove about 3 ul of the re suspended sample and spot on a glass microscope slide for cell examination Stain with Christmas Tree Stain refer to the CB Forensic Laboratory Serology Page 42 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 SOP Staining of Spermatozoa Kernechtrot Picroindigocarmine Steps and D spin only F remove supernatant G H may be repeated if nucleated or non nucleated non sperm cells are detected in the cell examination To the tube containing the washed pellet add 150 ul Tris EDTA NaCl 50 ul 20 Sarkosyl 7 ul 1M DTT 150 ul H20 and 4 ul of 10 mg ml Proteinase Close the tube caps and vortex for 1 second and spin in a microcentrifuge for 2 seconds to force all fluid and material to the bottom of the tubes Incubate at 56 C for a minimum of 2 hours To the tube containing the cell pellet and to the tube containing the female fraction add 300 ul phenol chloroform isoamyl alcohol Vortex low speed both tubes briefly to attain a milky emulsion Spin the tubes in a microcentrifuge for 3 minutes Note If necessary additional organic extractions can be
30. excluded Page 186 of 255 Appendix B Terms and Definitions for CODIS DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 SDIS State DNA Index System The centralized system of DNA identification records contributed to the state by all local participating laboratories Suspect An individual whose identity is known to the police and who is suspected to be the perpetrator of a crime Uploading The movement of DNA profiles between systems at different levels Page 187 of 255 Appendix B Terms and Definitions for CODIS DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 This Page Intentionally Left Blank Page 188 of 255 Appendix B Terms and Definitions for CODIS DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 APPENDIX C RESOURCES GENERAL RESOURCES The procedures described here are derived from a variety of sources Portions of the protocol come directly from some of the references cited below Budowle B Guisti A M Wayne J S Baechtel S Fourney R M Adams D E Presley L Deadman H A and Monson 1 Fixed Bin Analysis for Statistical Evaluation of Continuous Distributions of Allelic Data from VNTR Loci for Use in Forensic Comparisons Amer J Hum Genet 48 1991 841 55 National Research Council The Evaluation of Forensic DNA
31. ferrule on the right side of the pump block 3 Thread the capillary through the center of the ferrule 4 Tighten the ferrule in the block As the ferrule begins to seat adjust the end of the capillary so that it is positioned directly below the opening of the glass syringe With buffer valve closed push polymer down into block Allow to flow into capillary opening Finger tighten the ferrule to secure the capillary 5 Open the laser detector door and position the capillary in the vertical groove of the detector Position the center portion of the capillary detection window in the laser detector window Page 103 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 6 Close the laser detector door 7 Thread the other end of the capillary through the capillary hole in the electrode thumbscrew until it extends approximately 0 5 mm beyond the electrode 8 Secure the capillary in place by taping it to the heat plate above the laser detector door and above the electrode 9 Close the heat plate door NOTE The capillary injection counter must be reset each time a new capillary is installed M Fill the Buffer Reservoirs 1 Dilute 1 5 of 10X Genetic Analyzer Buffer with EDTA up to 15 makes a 1X solution with filter purified water NOTE A larger volume can be made if desired The 1X solution can be stored at 4 C for up to 2 weeks 2 Fill the anode buffer r
32. syringe Pull plunger so that the tip is drawn to the 0 8 to 1 0 ml mark Remove air bubbles from the tip and slowly expel the POP 4 through the syringe body and finally expel the POP 4 onto a Kimwipe or waste container Filling the syringe 1 Fill the 1 0 ml syringe to a maximum of 0 8 ml of POP 4 polymer NOTE The following formula can be used as a guideline for the approximate amount of polymer to sample 2 Sample x 6 ul POP 4 200 ul POP 4 NOTE Polymer that has been on the instrument for more than 72 hours should not be used for analysis Do not return the unused polymer to the original bottle 3 Rinse the outside of the syringe with distilled water and dry with a Kimwipe to remove excess polymer 4 Remove any air bubbles by inverting the syringe and pushing out a small amount of polymer NOTE It is critical that all air bubbles be expelled from the syringe Cleaning the Pump Block 1 Within the Manual Control window select Syringe Home from the Function pop up menu Select Buffer Valve Open press Execute Unscrew the glass syringe Remove any previously installed capillary Page 102 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 NOTE If the capillary has been used for less than 300 injections it may be stored with both ends in 1x Buffer or ultrapure H20 4 Grasp the block with both hands and pull straight out 5 Rinse t
33. 0 2723 0 3149 0 2813 12 0 1866 0 3391 0 2861 0 2542 13 0 1651 0 1634 0 1034 0 1625 14 0 0120 0 0322 0 0240 0 0354 15 0 0120 0 0124 0 0120 0 0104 gt 15 0 0120 0 0124 0 0120 0 0104 Page 208 of 255 Appendix D Allele Frequencies CBI DNA Analysis SOP Vers 2 1 06 13 02 4 43 PM CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 WORKSHEETS Page 209 of 255 Worksheets CBI DNA Analysis SOP Vers 2 1 06 13 02 4 43 PM CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 APPENDIX E DNA QUANTITATION MODEL Case Number s Analyst Date Unless otherwise stated 5 ul of the DNA standard was added to the tube ND Not detected gt Greater than lt Less than Spotting Solution QuantiBlot Wash Solution Biodyne B Membrane 1X Citrate Buffer QuantiBlot Kit 30 Hydrogen Peroxide PreWetting Solution 3 Hydrogen Peroxide Hybridization Solution Tetramethyl Benzidine Page 210 of 255 Appendix E DNA Quantitation Model CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 APPENDIX F DNA SuMMARY MODEL EXHIBIT DQA1 LDLR GYPA HBGG D7S8 GC 01580 211 255 DNA Summary CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002
34. 1 JUNE 2002 the capillary To make fine adjustments press the shift key while clicking the direction arrow The end of the capillary must be centered on and nearly touch the calibration point When complete select Done From the menu select Manual Control and select Autosampler to Position from the pop up menu 1 in the value box and select Execute Then select Autosampler Up and Execute until the capillary is inside the 1x buffer Prime the pump block 1 2 3 Select Buffer Valve Close from the Function pop up menu and click Execute Open the waste valve below the syringe by partly unscrewing it Push down on the syringe until the valve space is filled with polymer and then tighten the valve making sure no air is in the pump block space above the waste valve Open the valve to the left of the syringe and push polymer through to that valve region Push down on the syringe until the valve space is filled with polymer and then tighten this valve making sure no air is in the pump block space above the waste valve Select Buffer Valve Open from the Function pop up menu and click Execute Press down on the syringe in order to push polymer through the remainder of the block channel Observe as the bubbles travel through and out of the block and a drop of polymer falls through the anode buffer Select Buffer Valve Close from the Function pop up menu and click Execute There should be no bubbles in the block channe
35. 149 RFU can be incorporated in this assessment Page 116 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 Major Minor Contributors A sample can be considered to consist of a major minor contributor mixture if there is a distinct contrast in peak intensities between alleles and the alleles contributing the largest peak height values satisfy the conditions of a single source specimen All loci for which a result was obtained must be considered when making this determination Interpretation of the major contributor will be restricted to those major component peaks greater than or equal to 150 RFU Providing these conditions exist a genotype frequency will be reported for the major contributor The genotype of the minor contributor can be determined if the peaks once the major contributor s alleles have been accounted for satisfy the conditions of a single source specimen All loci must be considered when making this determination and there must be a clear distinction of the minor component from the major component at all loci all 9 if Profiler Plus and all 13 if both Profiler Plus and Cofiler utilized Interpretation of the minor contributor will be restricted to those minor component peaks greater than or equal to 150 RFU Providing these conditions exist the minor component will be considered defined and a genotype frequency will be reported for the minor contributor Mixtures
36. 1M SODIUM ACETATE pH 5 2 100 ml Dissolve 13 6 CH3COONae3H20 80 ml ultrapure water Adjust to pH 5 2 by adding glacial acetic acid approximately 2 ml Adjust the final volume with ultrapure water to 100 ml Sterilize by autoclaving Store at room temperature 5 M SODIUM CHLORIDE 1 L Dissolve 292 2 NaCl in 800 ml ultrapure water Adjust final volume with ultrapure water to 1 liter Sterilize by autoclaving Store at room temperature 20 w v SDS SODIUM DODECYL SULFATE IL CAUTION Wear protective mask when weighing SDS Slowly dissolve 200 g electrophoresis grade ultra pure sodium dodeoyl sulfate SDS in 800 ml sterile ultrapure water Warming e g in a 37 C water bath may be required to dissolve solids completely Adjust volume to 1L with sterile ultrapure water and mix thoroughly Store at room temperature SPOTTING SOLUTION 0 4N NaOH 25mM EDTA 0 00008 BROMOTHYMOL BLUE 75 ml Add 3 ml 10N NaOH 3 75 ml 0 5M EDTA and 150 ul 0 0496 Bromothymol Blue provided in QuantiBlot amp Kit to 68 ml ultrapure water and mix thoroughly Spotting Solution is stable for three months when stored at room temperature 20X SSPE BUFFER 3 6M NaCl 200mM NaH2PO eH20 20mM EDTA pH 7 4 1L Dissolve 14 8 g disodium ethylenediaminetetraacetic acid dihydrate in 1600 ml ultrapure water Adjust the pH to 6 0 0 2 with 10N NaOH solution Add 420 g sodium chloride NaCl and 55 2 g sodium phosphate monobasic monohydrat
37. 5 seconds VAGINAL SWABS OR SEMEN STAINS Vaginal swabs or semen stains should be air dried and stored at 20 C until used A Using a clean surface dissect the swab material from the applicator stick or cut a portion of the stained material and place it into a 2 2 ml microcentrifuge tube B To the sample add 400 Tris EDTA NaCI 25 ul 20 Sarkosyl 75 ul and 2 ul of 10 mg ml Proteinase K Vortex for 1 second and spin in a microcentrifuge for 2 seconds to force the material into the extraction fluid C Incubate at 379C for 2 hours D Transfer the swab material into a Spin X basket insert Place the basket insert into the tube containing the stain extract Spin in a microcentrifuge at maximum speed for 5 minutes E Remove the basket insert from the extract tube Remove the extracted material from the basket place in a clean microfuge tube and freeze if required Otherwise discard F While being very careful to not disturb any pelleted material remove the supernatant fluid from the extract and place it into a new labeled tube THIS SUPERNATANT IS THE NON SPERM EXTRACTION ANALYSIS OF THE NON SPERM FRACTION RESUMES AT STEP K THE PELLET REMAINING IN THE TUBE IS THE CELL PELLET G Wash the cell pellet by re suspending it in 500 ul sperm wash buffer vortexing the suspension briefly and spinning the tube in a microcentrifuge at maximum speed for 5 minutes Remove and discard the supernatant fluid being careful not to
38. 6 Signals obtained for non Water bath temperature too Water bath temperature human DNA samples low should be 50 C 19 SSPE concentration too Check that the 20X SSPE high in QuantiBlot Wash solution and the Solution QuantiBlot Wash Solu tion were prepared cor rectly DNA from primate species may give signals similar to those obtained from equivalent amounts of human DNA In Roche Molecular Systems RMS laboratories 30 ng to 300 ng quantities of non primate DNA samples result in either no signals or signals that are less than or equal to the signal obtained for 0 15 ng of human DNA The following non primate DNA samples have been tested in RMS laboratories E coli yeast dog cat mouse rat pig cow chicken fish and turkey Page 65 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 REFERENCES Walsh P S Varlaro J and Reynolds R A Rapid Chemiluminescent Method for Quantitation of Human DNA Nucleic Acids Research 20 1992 5061 5 Waye J S and Willard H F Structure Organization and Sequence of Alpha Satellite DNA from Human Chromosome 17 Evidence for Evolution by Unequal Crossing over and an Ancestral Pentamer Repeat Shared with the Human X Chro mosome Molecular and Cellular Biology 6 1986 3156 65 Whitehead T P Thorpe G H G Carter T J N Groucutt C and Kricka L J Enhanced Luminescence Procedure for Sensitive Determi
39. A Costar spin basket may be utilized for the agitation of the cellular component from the substrate See page 13 E OPTIONAL Spin microcentrifuge for 2 minutes at 10 000 to 15 000 F Without disturbing the pellet remove and discard all but 50 ul of the supernatant using a microtip disposable transfer pipette Re suspend the pellet in the remaining 50 ul by stirring with a sterile pipette tip G OPTIONAL Remove about 3 ul of the re suspended sample and spot on a glass microscope slide for examination Perform Christmas Tree Stain Serology SOP Staining of Spermatozoa Kernechtrot Picroindigocarmine NOTE If epithelial cells are detected follow the post coital protocol beginning with Step I If not detected proceed as follows To the approximately 50 ul re suspended cell pellet add 5 Chelex to a final volume of 200 ul Add 2 yl of 10 mg ml Proteinase and 7 ul of 1M DTT gently Incubate at 56 C for 30 to 60 minutes Vortex at high speed for 5 to 10 seconds Spin in a microcentrifuge for 10 to 20 seconds at 10 000 to 15 000 x g Follow protocol for Blood bloodstains beginning with Step H ORAL SWABS WHICH CONTAIN SPERMATOZOA A Dissect swab Use a clean cutting surface for each different sample Page 36 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 Pipet 150 to 500 ul of PBS into a sterile 1 5 ml microcentrifuge tube Add swab
40. AND ABI 3100 The ABI 377 ABI 3107 and ABI 3100 are maintained Each instrument is verified annually with a NIST traceable standard MATERIAL SAFETY DATA SHEETS MSDS MSDS have been obtained for all chemicals handled in the DNA laboratory The MSDS are maintained in a binder and are updated as needed Each analyst is aware of the binder s existence and location LOGBOOKS FOR CHEMICALS COMMERCIAL KITS IN HOUSE REAGENTS AND CRITICAL REAGENTS Logbooks of lot numbers expiration dates and verifications of lots for chemicals commercial kits for DNA typing in house reagents and critical reagents are kept in a readily accessible area RECORDS OF EQUIPMENT TEMPERATURES MAINTENANCE AND CALIBRATION Records of equipment temperature maintenance and calibration are kept in a logbook in a readily accessible area Page 20 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 PROFICIENCY TESTING Proficiency testing is used to demonstrate the quality of performance in the DNA Laboratory and serves as a mechanism for critical self evaluation This is accomplished by the analysis and reporting of results from appropriate biological specimens submitted to the Laboratory from an outside source of proficiency tests and also from specimens prepared in house All specimens submitted as part of a proficiency test must be analyzed at all 13 CODIS STR loci Additional loci can be a
41. C T and Budowle B Validation Studies on the Analysis of the HLA DQa Locus Using the Polymerase Chain Reaction Journal of Forensic Sciences 36 1991 1633 48 Comey C T Budowle B Adams D E Baumstark A L Lindsey J A and Presley L A PCR Amplification and Typing of the HLA DQa Gene in Forensic Samples Journal of Forensic Sciences 38 1993 239 49 Sullivan K et al Characterization of HLA DQA1 for Forensic Purposes Allele and Genotype Frequencies in British Caucasian Afro Caribbean and Asian Populations International Journal of Legal Medicine 105 1992 17 20 Saiki R K Walsh P S Levenson C H and Erlich H A Genetic Analysis of Amplified DNA with Immobilized Sequence specific Oligonucleotide Probes Proceedings of the National Academy of Sciences USA 86 1989 6230 4 Page 89 of 255 CBI DNA Analysis SOP Vers 2 1 20 21 22 23 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 Herrin G Jr Fildes N and Reynolds R Evaluation of the AmpliType PM DNA Test System on Forensic Case Samples Journal of Forensic Sciences 39 1994 1247 53 Budowle B Lindsey J A DeCou J A Koons B W Giusti and Comey C T Validation and Population Studies of the loci LDLR GYPA HBGG 0758 and GC PM loci HLA DQa Using a Multiplex Amplification and Typing Procedure Journal of Forensic Sciences 40 1995 45 50 Roy an
42. DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 5 Formamide lot 6 Cases included in the run Any other additional comments associated with the run may be added to the note section Fill out an Injection List 1 Select New from the ABI collection File menu Click on the GeneScan Injection List icon 2 In the Injection List window which appears select the appropriate Sample Sheet pop up menu 3 Insure that the correct Module was inserted into the Injection List The correct parameters for Module GS STR POP4 1ml F are a Inj Secs 5 b Inj kV 15 0 Run kV 15 0 and d Run C 60 NOTE If it is necessary to re inject a sample due to off scale data the injection time may be decreased Set the run time to insure that the necessary base pair peaks captured e g 24 30 minutes to obtain the 75 base pair to 400 base pair peaks of the GS500 NOTE The time necessary to obtain the 400 base pair peak can be set as a default module for the instrument f For additional injections highlight the Inj and select Command l from the keyboard Highlight the sample row and from the pop up menu select the desired sample g It is recommended that the first two injections should be from the BLANK tube Run a Seq Fill Capillary with one injection especially if it is a new run and or new polymer was placed in the syringe If desired a third blank can be run at the start of the run h It is recomm
43. Date Locus Allele Peak Height Interpretation and Comments rise to observed mixed profiles A major B minor 0351358 AMELOG 1 CSF1PO D7S820 Page 218 of 255 Appendix L Mixture Analysis Worksheet Cofiler CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 APPENDIX M DNA STOCK SOLUTIONS Loc 8 uG uL SERUM ALBUMIN BSA MODEL Solution Preparation Before starting UV treat all equipment and glassware Add 8 g BSA per every 1L 0 8 g per every 100 ml autoclaved ultrapure H2O Mix thoroughly Sterile filter through a Nalgene 0 2 or 0 45 micron filter unit Aliquot into sterile 15 ml tubes Aliquot one 15 ml tube into sterile 1 5 ml Sarstedt tubes as needed Store at 20 C BSA Lot Preparer Date Prepared Amount BSA2 Page 219 of 255 Appendix M DNA Stock Solutions Log 8 uG uL Bovine Serum Albumin BSA CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 APPENDIX N DNA STOCK SOLUTIONS Loc 10X CITRATE BUFFER MODEL 1M Sopium CITRATE 5 0 2 L Solution Preparation Dissolve 368 g trisodium citrate dihydrate 507 e 2H2O in 1600 ml ultrapure water Adjust pH to 5 0 0 2 using approximately 60 g citric acid monohydrat
44. Evidence Washington National Academy Press 1996 PCR Based Typing Protocols Laboratory July 1 1996 AMELOGENIN Akane A Shiono H Matsubara K Nakohori Y Seki A Nagafuchi S Yamada Nakagome Y Sex Identification of Forensic Specimens by Polymerase Chain Reaction PCR Two Alternative Methods Forensic Sci Int 49 1991 81 8 Akane A Seki A Shiono H Nakamura H Hasegawa M Kagawa M Matsubara K Nakahori y Nagafuchi S Nakagome Y Sex Determination of Forensic Samples by Dual PCR Amplification of an X Y Homologous Gene Forens Sci Int 52 1992 143 8 Buel et al PCR Amplification of Animal DNA with Human X Y Amelogenin Primers Used in Gender Determination J Forens Sci 40 1995 641 4 Casarino L De Stefano F Mannucci A Canale M HLA DQA1 and Amelogenin Coamplifications A Handy Tool for Identification J Forens Sci 40 1995 456 8 Deutsch D Structure and Function of Enamel Gene Products The Anatomical Record 224 1989 189 210 Kasi K Nakamura Y and White R Amplification of a Variable Number of Tandem Repeats VNTR Locus pMCT118 by the Polymerase Chain Reaction PCR and Its Application to Forensic Science J Forens Sci 35 1990 1196 200 Lagerstrom et al Mapping of the Gene for S linked Amelogenesis Imperfecta by Linkage Analysis Amer J Hum Genet 46 120 5 Page 189 of 255 Appendi
45. For alleles differing by two repeats the stutter peak from the larger allele may overlap the trailing shoulder of the smaller allele and therefore exhibit an increased stutter percentage This will not be the case if the smaller allele goes to the baseline before reaching the stutter peak C Stutter peaks overlapping an area of elevated baseline may exhibit increased percentages The analyst may choose to apply a different matrix and re analyze the sample with GeneScan software D The presence of a minor component as in mixture at the same location as a stutter peak NON TEMPLATE NUCLEOTIDE ADDITION A ADDITION AmpliTaq Gold DNA Polymerase can add an additional nucleotide to the 3 ends of double stranded PCR product This addition creates a species n 1 nucleotide in length The n 1 species is considered a true allele In some instances most notably in situations of sample overload and possibly suboptimal PCR conditions there can be incomplete addition n and two species will result that differ by one base pair n and n 1 The following considerations should be made in mixture interpretation to determine if a peak in the n position is a true peak an allele or possibly both A Determine if all the alleles are accounted for B Determine at which loci the n peaks are present vWA 0351358 THO1 seem to present this situation most readily C Determine if common alleles are known to exist at these loci in the n position
46. Peak height ratios of heterozygote alleles are defined as the percentage of the lower peak s height to the higher peak s height expressed as a ratio Peak heights below 70 may possibly indicate the presence of a mixture However a single source sample may have a peak height ratio below the 70 associated with a heterozygous allele at a given locus Careful examination of all loci with the indication of other peak height ratios lt 70 and or the presence at a given locus of three or more alleles will aid in the interpretation of the sample Depending on the sample source the locus in question the number of loci affected and the percent disparity between alleles the sample may need to be reamplified and typed Degraded DNA inhibitors low amount of target DNA or primer mismatches may be causes for the peak height ratio imbalance In cases where not all of the loci in the kit are developed a partial DNA profile is obtained and so noted in the report Within a single source sample genotypes generated for 0351358 075820 in the Profiler Plus and Cofiler amplification and typing systems must be concordant Artificial peaks within the analysis range other than target peaks may be detected on the electropherograms These extra peaks may be caused by stutter incomplete A addition pull up or spikes STUTTER Stutter is predominantly a minor product peak four base pairs shorter n 4 than the corresponding allele peak n It is al
47. Probe to the Hybridization Solution Place the lid on the tray Rotate in a 50 C 1 C water bath 50 to 60 rpm for 20 minutes 2 minutes Pour off the solution 3 Rinse the membrane briefly 100 pre warmed QuantiBlot Wash Solution by rocking the tray for several seconds Pour off the solution 4 Stringent Wash Conjugation add 30 ml pre warmed QuantiBlot Wash Solution to the Hybridization Tray Tilt the tray to one side and add 180 yl Enzyme Conjugate HRP SA to the 30 ml Wash Solution Place the lid on the tray Rotate in a 50 C 1 water bath 50 to 60 rpm for 10 minutes 1 minute Pour off the solution 5 Rinse the membrane thoroughly for 1 minute in 100 ml pre warmed QuantiBlot Wash Solution by rocking the tray or rotating it on an orbital shaker 100 to 125 rpm at room temperature Pour off the solution Rinse again for 1 minute Pour off the solution Page 60 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 6 Wash the membrane by adding 100 pre warmed QuantiBlot Wash Solution to the tray Place the lid on the tray Rotate at room temperature on an orbital shaker 100 to 125 rpm at room temperature for 15 minutes Pour off the solution re Rinse the membrane briefly in 100 ml Citrate Buffer by rocking the tray Pour off the solution Colorimetric Detection 1 Prepare the Color Development Solution not more than 10 minutes before us
48. RFU in unanalyzed data with the reviewer s approval for CODIS upload Alternatively the sample may be diluted and rerun or reamplified and retyped using a smaller amount of target DNA OFF LADDER ALLELES Peaks not aligning with those in the allelic ladders may be detected both within and outside of the range of the allelic ladders The Genotyper s software will accurately label many of the alleles not present in the allelic ladders however manual genotyping may be used to determine a genotype where Genotyper does not make the assignment Manual genotypes are assigned using the base pair designations calculated by the GeneScan software Whenever an off ladder allele is considered a true off ladder allele a variant allele rather than an off ladder allele due to spikes pull up split peaks etc it Page 154 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 must be confirmed by retyping if it conforms to the same overall guidelines described in the Evaluation of STR Data section Off ladder alleles OLA that fall between alleles within the ladder will be designated in accordance with guidelines of the International Society of Forensic Haemogenetics OLA calls are first converted to sizes in base pairs then compared to the size of the appropriate ladder alleles and the allelic designation determined If the OLA is not a perfect repeat but rather varies by 1 2 or 3 base pairs from a
49. Remove labels from stutter peaks Build a table containing genotypes for all samples Note If the Sample Info or Color Info field in the sample sheet plate record was not completed before initiating a run it is possible to add this information once the data is in the Genotyper application 1 Under the View menu choose Show Dye Lanes Window 2 Select the first sample by clicking on the row 3 Select the Sample Info box at the top of the window and type the sample description 4 Repeat the above steps for every sample in the Dye Lanes list Enter the same sample description for each of the dye colors for a single sample UsiNG TEMPLATE FILE NOTE The template file is a Stationary Pad which means that a new document is created each time it is opened The original template file is not overwritten A Double click on the template file of interest to simultaneously open the Genotyper application and the template file B Import GeneScan project by selecting Import from GS File Select project file from the appropriate Run Folder by double clicking on it Click finish to import file into Genotyper Save Genotyper file as GTdateanalD PP CO C From the Macro list at the bottom left of the window select Check GS 500 Double click on this macro or use command listed to execute the macro In the Plot window that appears scroll through each sample and verify the presence of both the 75 and 400 bp peaks Confirm c
50. Vers 2 1 gt 30 0 0139 0 0128 0 0123 0 0131 D8S1179 Allele Pesca Caucasian 2 1 mE N 180 UNE T9 N 203 191 lt 8 0 0139 0 0128 0 0123 0 0131 8 0 0139 0 0179 0 0123 0 0157 9 0 0139 0 0128 0 0123 0 0131 10 0 0250 0 1020 0 0936 0 0969 11 0 0361 0 0587 0 0616 0 0524 12 0 1083 0 1454 0 1207 0 1073 13 0 2222 0 3393 0 3251 0 3560 14 0 3333 0 2015 0 2463 0 2120 15 0 2139 0 1097 0 1158 0 1204 16 0 0444 0 0128 0 0246 0 0262 17 0 0139 0 0128 0 0123 0 0131 gt 17 0 0139 0 0128 0 0123 0 0131 200 255 06 13 02 4 43 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 ALLELE FREQUENCIES CONTINUED D21S11 African Southwest Southeast Allele American mE N 179 N 198 N 203 N 191 lt 24 2 0 0140 0 0128 0 0123 0 0131 24 2 0 0140 0 0128 0 0123 0 0131 24 3 0 0140 0 0128 0 0123 0 0131 26 0 0140 0 0128 0 0123 0 0131 27 0 0615 0 0459 0 0123 0 0183 28 0 2151 0 1658 0 0690 0 1257 29 0 1899 0 1811 0 2044 0 2408 29 2 0 0140 0 0128 0 0123 0 0131 30 0 1788 0 2321 0 3301 0 2382 30 2 0 0140 0 0383 0 0320 0 0340 30 3 0 0140 0 0128 0 0123 0 0131 31 0 0922 0 0714 0 0690 0 0759 31 2 0 0754 0 0995 0 0862 0 0838 32 0 0140 0 0153 0 0123 0 0131 32 1 0 0140 0 0128 0 0123 0 0131 32 2 0 0698 0 1122 0 1355 0 1152 33 0 0140 0 0128 0 0123 0 0131 33 2 0 0335 0 0306 0 0419 0 0367 34 0 0140 0 0128 0 0
51. amplification Sequence microvariants can affect the amount of stutter e g a lower amount of stutter is produced from alleles with increased sequence variation between repeats Database samples are run with a 20 stutter filter using KAZAM 20 in Genotyper Any sample giving stutter greater than the 2096 should be closely examined and using the professional judgment of the analyst determined to be either stutter or an actual peak The reviewing analyst may help in this call keeping in mind that it is always possible to rerun or re amplify the sample to help in this process Stutter peaks may be elevated above established thresholds by the following Off scale raw data RFU greater than 5000 If the stutter peak is greater than the maximum allowed and the primary peak is above 3000 RFU and or has been labeled off scale the analyst should interpret the results with caution The sample may be diluted and re run to resolve the issue For alleles differing by two repeats the stutter peak from the larger allele may overlap the trailing shoulder of the smaller allele and therefore exhibit an increased stutter percentage This will not be the case if the smaller allele goes to the baseline before reaching the stutter peak e Stutter peaks overlapping an area of elevated baseline may exhibit increased percentages The analyst may choose to apply a different matrix and re analyze the sample with GeneScan software NON TEMPLATE NUCLEOTIDE ADD
52. and Reynolds R Sequence Analysis and Characterization of Stutter Products at the Tetranucleotide Repeat Locus Nucleic Acid Res 24 1996 2807 12 Weber J and May P Abundant Class of Human DNA Polymorphisms Which Can Be Typed Using the Polymerase Chain Reaction Am J Hum Genet 44 1989 388 96 Ziegle J S Su Y Corcoran K P Nie L Mayrand E Hoff L B McBride L J Kronick M N and Diehl S R Application of Automated DNA Sizing Technology for Genotyping Microsatellite Loci Genomics 14 1026 31 CODIS CODIS Local Users Manual U S Department of Justice Federal Bureau of Investigation CODIS NDIS Procedure Manual U S Department of Justice Federal Bureau of Investigation CODIS State Users Manual U S Department of Justice Federal Bureau of Investigation January 1999 CODIS Training Manual U S Department of Justice Federal Bureau of Investigation CODIS v4 6 Import v1 7 Users Manual Page 195 of 255 Appendix C Resources CBI DNA Analysis SOP Vers 2 1 06 13 02 4 43 PM CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 This Page Intentionally Left Blank Page 196 of 255 Appendix C Resources CBI DNA Analysis SOP Vers 2 1 06 13 02 4 43 PM CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 APPENDIX D ALLELE FREQUENCIES D3S1358 African Southwest Southeast Allele American
53. and created through individual laboratory validation studies run prior to DNA database analysis Each laboratory will use the Run Module shown to work best in that laboratory The Run Module denotes the capillary length polymer used in collection and the injection time 6 Select an Analysis Module GS500Analysis gsp is typically used for fragment analysis using a 500 basepair internal size standard LIZ or ROX To inject a set of 16 wells more than one time fill in more than one Run Module column and its corresponding Analysis Module Any unused wells may be filled with internal size standard and formamide and labeled as such Any set of 16 wells not used should be deleted from the plate record T Name the plate record The plate record name is how the 3100 collection software will recognize the plate and the information regarding the samples on that plate The name of the plate should include the date analyst initials and amplification kit used i e mmddyyanalD If more than one plate is run on a single date then after the date the letter A or B may be used to designate the plate location on the 3100 instrument i e mmddyyAanalD for plate in the A position 8 Save the plate record The method used to create the plate record will determine how to save the plate record If it was created using the 3100 collection software clicking the OK button will save the plate and bring it up in the software as pending If using the Load
54. be entered into CODIS as a heterozygote If the peak height ratio is less than 35 no alleles for that locus may be entered into CODIS unless approved by the Technical Leader and CODIS Administrator TRI ALLELIC PROFILES Database samples with three alleles at a single locus may occur The sample is rerun or reamplified to confirm the type All three alleles are entered into CODIS This must be done using the keyboard since the software packages cannot enter three alleles for a single locus Disagreements between analysts regarding interpretation may occur analysts may first consult with the Technical Leader If the allele call s remain unresolved the allele under dispute will not be entered into CODIS Page 155 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 SPILLOVER AND LEAKAGE ABI 377 Database samples are known to be from a single donor Therefore in the interest of throughput database samples may be loaded in adjacent lanes of a gel During loading of a gel there is the possibility of a small amount of sample either spilling over into an adjacent lane or leaking under the sharkstooth comb into the next lane Interpretation is possible if the amount of extraneous sample is small Spillover leakage must be less than 10 of the adjacent sample peak heights for the profiles to be acceptable Samples viewed as acceptable by these standards may be rerun at t
55. case tables GT dateaiPP or COD50 5010 IBM date All case data will be stored associated with the month and date of the run That folder will be duplicated onto a write protected CD One CD will be stored in the lab from which the data originated and the second will be stored as follows Denver to Pueblo Pueblo to Montrose Montrose to Denver Page 121 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 REPORT WRITING CONTENTS OF PCR REPORTS All PCR reports shall contain date of submission and completion laboratory case number agency case number name and ORI of submitting agency item number and description of evidence list of loci analyzed summary of the test results conclusions Interpretative statement either quantitative or qualitative statement regarding disposition of evidence examiner designation initials and page numbering and signature and title of the person accepting responsibility for the content of the report GENERAL CATEGORIES OF TESTING CONCLUSIONS Exclusion The genetic information detected for the samples under comparison is different and could not have originated from a common source Inclusion match The genetic information detected for the samples under comparison is the same and the possibility of originating from a common source cannot be excluded Statistical information is generally included for probative comparisons which re
56. cause artifactual peaks Most artifactual peaks can be shown to be false by re injection of the sample The peaks fail to show in the same bp size location Spikes are one of the most common anomalies and can occur in one two three or all four colors Artifactual peaks occurring in all four colors are the easiest to diagnose as they will be the same size and of similar height within an order of magnitude of each other they do not need to be re injected if they do not interfere with the interpretation of the sample Artifactual peaks occurring in less than four colors should be interpreted with caution especially single color spikes and the analyst may choose to re inject to insure a clear interpretation Generally spikes are thin peaks with variable peak heights often having the same scan number OFF SCALE DATA Multicomponent analysis of off scale data may result in raised baselines excessive pull up of one or more colors under the off scale peaks or unnaturally high stutter peak ratios Off scale data may still be interpreted with caution Samples with off scale data 8100 RFU in unanalyzed data may be re injected with less time 2 3 seconds diluted and retyped or in some cases re amplify and type the sample using a smaller amount of target DNA OFF LADDER ALLELES Peaks not aligning with those in the allelic ladders may be detected both within and outside of the range of the allelic ladders The Genotyper software will accurate
57. choose NEW from the File menu 2 Select Insert from the table menu and open the appropriate CODIS GT PP file The PP profiles for the run will be imported 3 Select Insert again and open the corresponding CODIS GT CO file The CO profiles will be added to the PP profiles 4 Scroll through the profiles For each sample check that all alleles are present and that the results from D3 and D7 are concordant 5 For those samples with off ladder alleles replace OL allele with the appropriate allele value Page 158 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 C Complete the File Settings 1 To set the default go to Default Record under User Information This should read as follows Sample Info Untitled Sample 0 Category Convicted Offender Tissue Type Unknown Tissue Form Stain Population Unknown NOTE Default settings are set initially and should not be changed 2 Choose Untitled Settings from the Table menu and complete as follows User Lab CBI First Name Analyst Name Last Name Analyst Name User Initials AN User ID Your CODIS user name User email DNACOMM D Create a CMF common message format file 1 Choose Export CODIS from the Table menu This creates the DAT file 2 Save the DAT file with the run date and analysts initials mmddyyan DAT This DAT file will be imported into CODIS 3 Cho
58. color on the membrane will fade upon drying Page 61 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 Results Interpretation Results are interpreted by comparing the signal intensity of the DNA test sample to the signal intensity obtained for the DNA Standards The signal intensity for a sample reflects the total amount of DNA spotted on the membrane The seven DNA Standards represent the following quantities of DNA spotted on the membrane 10 5 2 5 1 25 0 625 0 3125 and 0 15625 ng See Table 1 The DNA Calibrators are used to provide DNA of a known concentration to verify that the DNA Standards were correctly diluted and are providing correct results for the test samples For example the DNA Calibrator 1 has a stock concentration of 0 7 ng ul Five ul of this control was added to 150 ul of Spotting Solution and the entire 155 ul was spotted on the membrane Thus 3 5 ng of this sample was spot ted on the membrane 0 7 ng l 5 ul 3 5 ng The signal obtained for this control sample should have an intensity that is between the 2 5 and 5 ng DNA Standards Likewise the DNA Calibrator 2 should have an intensity that is between the 0 3125 and 0 625 ng DNA Standards If not see the Troubleshooting Section The concentration of a DNA test sample is determined as follows 1 Determine the quantity of DNA test sample spotted on the membrane by comparing its signal intensity to th
59. decrease the possibility of contamination through repeated opening of the AmpliType PM DQA1 PCR Reaction Mix bottle Sections Precautions and PCR Amplification Protocols should be read before preparing AmpliType PM DQA1 PCR amplification reactions PRECAUTIONS The sensitivity of PCR allows minute quantities of DNA to be typed using the AmpliType PCR Amplification and Typing Kits Contamination of the samples by handling or by exposure to any other source of human DNA is an important concern Precautions should be taken to prevent the following three types of contamination 1 contamination with exogenous human genomic DNA 2 cross contamination between extracted DNA samples 3 carryover of PCR product from one amplification to the next 28 At a minimum the pre PCR amplification area must be separated from the post PCR amplification area PCR AMPLIFICATION PROTOCOLS The following protocols detail the PCR amplification and typing procedures specific for the AmpliType PCR Amplification and Typing Kits A dedicated area such as a biological hood or a separate room should be used for preparing AmpliType PCR amplification reactions All equipment and supplies used to prepare amplifications should be kept in this dedicated clean area at all times Do not use these items to handle amplified DNA or other potential sources of contaminating DNA Trace amounts of amplified DNA if carried over into other samples befo
60. filtrate cup and carefully invert the concentrator onto a labeled retentate cup Discard the filtrate cup Note Final recovery volumes following purification generally range from 20 ul for evidentiary question samples to 200 ul for reference known samples The final recovery volumes for the extraction reagent blanks for evidentiary samples and known samples are generally 100 ul and 200 ul respectively l Spin the assembly in a microcentrifuge at 2500 x g for 5 minutes Discard the concentrator Cap the retentate Estimate the quantity of DNA in the sample by slot blot hybridization After quantification the sample can be amplified Z qo ow T Store samples at 4 C or frozen Prior to the use of samples after storage they should be vortexed briefly and spun in a microcentrifuge for 5 seconds Page 44 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 ENVELOPE FLAPS OR STAMPS A Carefully open envelope flap or remove stamps using steam and clean tweezers Using a sterile cotton swab moistened in sterile filter purified water swab gummed envelope flap or stamp Cut cotton swab from stick and transfer the cotton to a 2 2 microcentrifuge tube Optionally gt of available dissected material can be placed into the extraction tube B To the sample add 300 ul organic stain extraction buffer 12 ul 1M DTT and 4 ul 10 mg ml Proteinase K solution Vortex for 1 seco
61. for analysis Number of Examinations Number of Specimens Hours per SOP Dept of Corrections Probation Division of Youth Corrections Number of Examinations Number of Specimens Page 183 of 255 Appendix A Analyst Activity AA Forms DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 TRV Technical ReView TP1 TRV AA Forms Counting Exams DNA Blood Hair or Tissue Specimen item STR 10 exam item PMDQa 5 exam item DNA Differential Extraction Specimen item STR 20 exam item PMDQa 5 exam item Page 184 of 255 Appendix A Analyst Activity AA Forms DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 APPENDIX B TERMS AND DEFINITIONS FOR CODIS Autosearcher A CODIS program which automatically searches all DNA profiles in user specified index against all profiles in or more other user specified indexes Candidate Match A possible match between two or more DNA profiles discovered by CODIS software Qualified DNA analysts must verify candidate matches CODIS COmbined DNA Index System The entire system of DNA indexes maintained at the national state and local levels CODIS Administrator Local or LDIS Responsible for overseeing the CODIS system Duties may include organization of CODIS materials installing software routine tape backup system QA routine local searches and overseeing
62. for the amplification of DNA except that no DNA is added TE Buffer is placed in the negative amplification blank in lieu of a DNA solution The negative amplification blank is processed through the amplification and electrophoretic typing procedures Positive Amplification Controls DNA from cell line 9947A is provided with the reagent kits This is used as a positive control for STRs and amelogenin to evaluate the performance of amplification and electrophoresis When the control specimen 9947A is amplified the STR loci must solely exhibit the correct genotype Additionally specimen 9947A is the female control for amelogenin and must exhibit a single band at the position corresponding with the size ladder band representative of the 106 bp peak from the X chromosome According to the AmpFISTR Identifiler PCR Amplification Kit Users Manual appropriate cycle number for the high quantity of DNA obtained from a 1 2 mm bloodstained FTA punch is 25 cycles Because the positive amplification control 9947A is not sufficiently amplified in the suggested 25 cycles a NIST traceable CBI internal control consisting of a blood standard of a known type spotted onto FTA paper can serve as the positive amplification control This internal control may differ for each laboratory The genotype for the CBI internal DNA controls is kept in the logbook with the results of the kit validations Page 128 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LAB
63. in the kit to the tube for the positive control 20 ul TE Buffer to the tube for the negative amplification blank SAMPLE PREPARATION CHELEX SAMPLES USING IDENTIFILER KIT A B Prepare a DNA TE dilution of each sample The final amount of input DNA is 0 5 to 1 25 ng diluted in TE to a final volume of 10 uL Determine the number of samples to be amplified Place the required number of amplification reaction tubes into a rack including tubes for the positive control reagent blank and negative amplification blank Treat the tubes with ultraviolet light for at least 20 minutes Page 132 of 255 CBI DNA Analysis SOP Vers 2 1 nmaoso G H FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 Prepare Master Mix according to Formulation instructions Dispense 15 ul of Master Mix into each reaction tube If using the 480 Thermal Cycler add one drop of mineral oil to each tube Open each sample tube add 10 ul of the appropriate DNA TE sample preparation and then close the tube Make sure to have only one tube open at a time Do not mix or vortex the tube Add 10 ul of the reagent blank to the tube for the reagent blank control Add 10 ul of the control DNA provided in the kit to the tube for the positive control Add 10 ul TE Buffer to the tube for the negative amplification blank SAMPLE PREPARATION WASHED SAMPLES FOR AND S amp S PAPER SAMPLES USING COFILER AND PROFILER PLUS KITS
64. it with autoclaved DI For most samples 2 to 5 ng is sufficient If the DNA sample contains degraded DNA it may be appropriate to add gt 10 ng of DNA Determine the number of samples to be amplified including positive and negative controls The Control DNA 1 provided in the kit shall be amplified each time the kit is used A negative control consisting of 40 ul of AmpliType PM DQA1 PCR Reaction Mix 40 of AmpliType PM DQA1 Primer Set 2 ul of Bovine Serum Albumin and 18 ul of autoclaved DI H2O shall also be included with each set of amplification reactions Transfer the PCR amplification reagents to the designated clean area Place the required number of reaction tubes containing 40 of aliquoted AmpliType PM DQA1 PCR Reaction Mix and 2 ul of Bovine Serum Albumin in a rack not used for the preparation of DNA or the handling of amplified DNA Label the reaction tubes Insure that the solution is at the bottom of each tube by spinning the tubes briefly in a microcentrifuge Open the caps with a clean microcentrifuge tube de capping tool this de capping tool should not have been used on tubes containing amplified or extracted DNA Avoid touching the inside surface of the tube caps Pipet 40 ul of the PM DQA1 Primer Set provided in the kit into each tube including control tubes using a sterile dedicated positive displacement pipette Page 70 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORAT
65. kg weight e g a lead ring on the covered tray to prevent the tray from sliding or floating Check the tray position and insure that water does not splash into the wells of the tray Adjust the water level if necessary If water must be added to the bath use water heated to 55 C 1 C K Replace the water bath cover to maintain the bath temperature at 55 C 1 C Hybridize the amplified samples to the DNA probe strips by incubating at 55 C for 15 minutes 2 minutes L Approximately 5 minutes before the end of the hybridization step prepare the Enzyme Conjugate Solution in a glass flask using the following equations to determine the volume of each component required Number 3 3 ml of Volume of Hybridization X Hybridization Solution Strips Solution Number 27 ul of Volume of Enzyme of Strips X Enzyme Conjugate Conjugate HRP SA HRP SA Mix the solution thoroughly and insure that the solids remain in solution Leave at room temperature 15 to 30 C until ready to use M After hybridization remove the tray Replace the water bath cover to maintain the temperature at 55 C 1 C Keep the water bath rotating between incubation steps N Aspirate the contents of each well from the labeled end of the strip while tilting the tray slightly Remove condensation from the tray lid with a clean lab wipe Use of paper towels to wipe the tray lid is not advised because some paper towels contain bleach NOTE PM DQA1
66. ladder allele then it will be designated as an integer of that variation For example if a green OL peak size is 238 89 and the 36 allele of D21S11 ladder is 236 82 bp then the peak will be designated 021511 36 2 Alleles smaller than the lowest molecular weight allele i e A will be designated lt A Alleles greater than the largest molecular weight allele i e B will be designated gt B The profile must be confirmed by retyping if it conforms to the same overall guidelines described in the Evaluation of STR Data section If upon evaluation of the profile the allele in question is at a position of an overlap between two loci then the sample should be analyzed using Profiler Plus and Cofiler Off ladder homozygous alleles are rare and must be interpreted with caution due to the possibility of allelic dropout due to primer binding site mutations Allelic balance across the profile should be examined If the peak heights of the off ladder homozygous allele and the heterozygous alleles are similar the off ladder homozygous allele should be considered an uninterpretable locus and not be entered into CODIS PEAK HEIGHT IMBALANCES Imbalances in peak height at a heterozygous locus may occur This may be a result of a primer binding site mutation Samples containing imbalances should be rerun or reamplified to confirm the profile If the peak height ratio at an unbalanced locus is consistent and is greater than or equal to 35 the profile may
67. ml tube is recommended when preparing Master Mix for up to 55 samples Be sure to include enough Master Mix for reagent blanks a positive control and a negative amplification blank Mix thoroughly by vortexing at medium speed for 5 seconds Spin the tube briefly in a microcentrifuge to remove any liquid from the cap Dispense 30 of Master Mix into each GeneAmp Thin Walled Reaction tube If using the 480 thermal cycler add one drop mineral oil supplied with kit to each of the tubes This step not necessary if using the 9700 thermal cycler To each forensic sample tube add 20 ul of sample DNA TE sample preparation insert the pipette tip below the oil layer if using the 480 thermal cycler After the addition of sample cap the tube before proceeding on to the next tube Do not mix or vortex the tube The final PCR reaction volume is 50 yl Add 20 yl of the reagent blank to a tube for the reagent blank control Add 20 ul of the diluted positive control DNA provided in the kit in a concentration consistent with the amount of DNA used in the forensic samples to a tube for the positive control 20 ul TE Buffer to a tube for the negative amplification blank Place the PCR tubes in the thermal cycler and start the program If using the 480 record the heat block position of each tube After the amplification process remove the tubes from the thermal cycler and store the amplified products protected from light The amplified
68. number profile or the most complete profile from the analysis will be entered i e if four bloodstains on a pair of jeans X1 3 1 through X1 3 4 yield the same DNA profile only X1 3 1 will be entered into LDIS Page 167 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 The following format for Specimen ID will be used for all CBI cases 1 Forensic Unknowns Exhibit Case i e 1D000000 There are to be no spaces or punctuation Profiles from cases will be entered using the following procedure 1 Log on to CODIS Local by entering you CODIS User ID and password Do not enter profiles under another analyst s name 2 Under PCR Analysis click on the PCR button This opens the Select Add PCR Specimen screen 3 Enter the Specimen ID in the Specimen ID blank Check the entry for accuracy 4 Select the applicable Specimen Category e g Forensic Unknown by scrolling through drop down menu then highlighting the selection This specifies the Index for that profile 5 Check that the analyst s name next to Assigned To is correct If not repeat the Log on process using the name of the analyst performing the profile entry Click on the Add button 7 Enter allele calls into the table under Reading Information Click on the blank box under each locus and enter the alleles for that locus separating the individual alleles with a comma Do not use spaces The first and
69. of the cellular component from the substrate G Without disturbing the pellet remove all but 50 ul or twice the volume of the pellet whichever is greater of the supernatant using a sterile Pasteur pipette or the tip of a sterile 1 ml disposable pipette and place into the tube containing the swab fabric This supernatant can be used for traditional serological analyses if necessary Re suspend the pellet in the remaining 50 ul by stirring it with a sterile pipette tip This pellet contains epithelial cells and sperm cells and is called the cell debris pellet H OPTIONAL Remove about 3 ul of the re suspended sample and spot on a glass microscope slide for examination Stain with Christmas Tree Stain refer to the CBI Forensic Laboratory Serology SOP Staining of Spermatozoa Kernechtrot Picroindigocarmine NOTE If epithelial cells are detected proceed with the differential lysis procedure beginning with Step I If no epithelial cells are observed the differential lysis procedure may be omitted and the sample may be processed beginning with Step Q l To the approximately 50 ul of re suspended cell debris add TE Buffer to a final volume of 200 ul Add 2 ul of 10 mg ml Proteinase K Mix gently Page 34 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 J Incubate at 37 C for about 1 hour to lyse epithelial cells but for no more than 2 hours to minimize lysi
70. products can be stored at 2 to 6 C for no more than 2 weeks For longer periods store the tubes at 15 to 20 C Page 100 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 STR TYPING BY CAPILLARY GEL ELECTROPHORESIS SETTING UP THE INSTRUMENT A Turn on the ABI PRISM 310 Genetic Analyzer B Restart the Power Macintosh computer Launch the ABI 310 Collection software if it is not already open Note It is not necessary to change the capillary for every run The maximum of injections per capillary is approximately 300 C Select Change Capillary from the Instrument menu to determine whether the capillary needs to be changed Select Cancel to close D Remove the POP 4 from the refrigerator to warm to room temperature If precipitate is present when the bottle is removed warming to room temperature and gentle mixing rotate slowly by hand carefully so as to not introduce air into the POP 4 V will remove the precipitate If the precipitate will not dissolve do not use the POP 4 E Home the instrument as follows 1 Under Windows menu select Manual Control 2 Select Syringe Home from the Function pop up menu and click Execute 3 Select Autosampler Home X Y Axis from the Function pop up menu and click Execute 4 Select Autosampler Home Z Axis from the Function pop up menu and click Execute F Installing a New Electrode If this procedure is not nec
71. results may be obtained outside of this range Page 98 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 AMPLIFICATION All PCR setup steps must be performed in the DNA Laboratory using hoods reagents and pipettors dedicated to this area The reagents required for the amplification of the thirteen core STR loci are included in two kits the AmpF STR Profiler Plus PCR Amplification Kit and the AmpF STR Cofiler PCR Amplification Kit For each amplification combine DNA template PCR reaction mix Primer set Profiler Plus or Cofiler and DNA polymerase AmpliTaq Gold in a reaction tube and subject this mixture to a series of controlled temperature changes in the Thermal Cycler Thermal Cycler Parameters The following parameters are used to amplify the AmpF STR Profiler Plus and Cofiler loci on the PE Biosystems thermal cyclers Turn on the Thermal Cycler and allow the instrument to warm up for a minimum of 1 hour Add one drop of oil 480 s only into each of the wells to be used Select the file for forensic STR analysis The parameters are listed below DNA Thermal Cycler 480 or 9700 Pre heat step cycle file 1 cycle 95 C 11 minutes Cycle step cycle file 28 cycles 94 C 1 minute 59 C 1 minute 72 C 1 minute Final extension step cycle file 1 cycle 60 C 90 minutes Hold temperature soak file 10 C indefinite Tubes can be left in the Thermal Cy
72. small pieces B Add 1 ml TE Buffer Incubate at room temperature for 15 to 30 minutes Mix occasionally by inversion or gentle vortexing C Spin in a microcentrifuge for 2 to 3 minutes at 10 000 to 15 000 x g D Carefully remove supernatant all but 20 to 30 ul and discard Leave the fabric substrate in the tube with the pellet Add 5 Chelex to a final volume of 200 ul Incubate 56 for 15 to 30 minutes or overnight Overnight is optimal Vortex at high speed for 5 to 10 seconds Spin in a microcentrifuge for 5 to 10 seconds at 10 000 to 15 000 x g H Incubate in a boiling water bath for 8 minutes l Vortex at high speed for 5 to 10 seconds J Spin in a microcentrifuge for 2 to 3 minutes at 10 000 to 15 000 x g K The sample is now ready for the PCR Amplification process L Store the remainder of the supernatant at 2 to 8 C or frozen To re use repeat Steps I through Page 38 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 HAIR M Handling hair with clean forceps examine the hair under a dissecting microscope for the presence of sheath material The hair may be placed on a clean piece of white paper Note possible presence of body fluids on hair N Wash the hair containing sheath material thoroughly to reduce surface dirt and contaminants by immersing the hair in sterile saline Follow this by either of the following cutting methods Al
73. t c t e om e ud MEAS 129 DNA THERMAL CYCLER 480 AND 9700 22 2 129 ABI PRISM 377 DNA 7 129 ABI PRISM 3100 GENETIC ANALYZER 129 DESKTOP GOMPUTER GS eene debut uci Gs s Ga GO D EN OR VG ak E RR 129 QUALITY CONTROL OF CRITICAL REAGENTS 129 PROFILER PLUS COFILER AND IDENTIFILER 129 HIP 130 EXTRACTION PROCEDU RE 130 QUANTIFICATION PROGEDURE lec 130 130 THERMAL CYCLER 8 130 DNA THERMAL CYCLER 480 9700 131 AMPLIFICATION MIX 131 SAMPLE PREPARATION CHELEX SAMPLES USING COFILER AND PROFILER PLUS KITS be A 132 SAMPLE PREPARATION CHELEX SAMPLES USING IDENTIFILER KIT 132 SAMPLE PREPARATION FTA WASHED SAMPLES FOR FTA AND S amp S PAPER SAMPLES USING COFILER AND PROFILER PLUS KITS 133 SAMPLE PREPARATION FTA WASHED SAMPLE FOR FTA AND S amp S PAPER SAMPLES USING IDENTIFILER KIT 2 2 133 PAMPEIFIGATIONS 22226 6 aiite 134 STR TYPING BY POLYACRYLAMIDE GEL ELECTROPHORESIS
74. the capillary has been used for less than 300 injections it may be stored with both ends in 1X Buffer 3 Grasp the block with both hands and pull straight out 4 Rinse the blocks valve tubing and ferrule thoroughly with warm tap water and then with filter purified water A plastic syringe with an adapter may be used for this process 5 Remove any excess water from inside and outside of the blocks using compressed air and Kimwipes Page 144 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 6 Verify that the gold electrode socket on the back of the pin valve block is dry and then replace the block Slide the U shaped end of the activator arm into the collar at the top of the plunger valve Make sure the activator arm lines up with the groove in the pin valve before pressing into place It may be necessary to move the activator arm up or down by selecting Pin Valve Open or Close from the Manual Control menu GENESCAN ANALYSIS All profiles generated from database samples are reviewed by two analysts The results are compared Any discrepancies must be resolved prior to CODIS entry The ABI 377 DNA Sequencer uses a Macintosh computer platform to collect raw data and also uses Genescan analysis software A matrix file is applied to the raw data to correct for spectral overlap and produce singular peaks of the four individual colors A size curve is created using an
75. the samples at 95 C for a minimum of 3 minutes and no longer than 5 minutes Place the entire 96 well plate into a 9700 programmed at 95 C NOTE Septa or Septa Strips melt at high temperatures Do not autoclave or re use septa 6 Snap cool on ice bath immediately for a minimum of 3 minutes Starting the run 1 Remove the plate from the ice bath and dry off any excess moisture from the plate prior to placement into the plate rack assembly Place the plate into the plate assembly Make sure the plate is centered on the rack by aligning holes on the upper assembly with the holes in the septa The assembly clicks together to assure it is locked NOTE To avoid electrical arcing it is imperative that all surfaces of the buffer and water reservoirs and the plate assembly be dry 2 Place the assembly onto the plate deck in the correct position A or B Firmly push down on the assembly to be sure it is seated completely onto the deck Close the doors on the 3100 instrument and wait for the instrument to home itself 3 On the computer link the plate record with the plate position A or B by highlighting the pending plate record and then clicking on the position to be used Wait while the computer links all records Click on the green arrow on the menu bar to start the run Page 143 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 Ending a run When a run is comple
76. thermal cycler and start the program If using the 480 Thermal Cycler record the heat block position of each tube This is not necessary when using the 9700 Thermal Cycler B After the amplification process remove the tubes from the thermal cycler and store the amplified products protected from light The tubes may be centifuged to get the PCR product to the bottom C The amplified products can be stored at 2 6 C for no more than 1 week For longer periods store the tubes at 15 to 20 C STR TYPING BY POLYACRYLAMIDE GEL ELECTROPHORESIS USING THE ABI 377 POURING A POLYACRYLAMIDE GEL A 36 cm polyacrylamide gel should be poured a minimum of 2 hours prior to setting up the 377 DNA Sequencer Gels may be stored wrapped at room temperature up to 24 hours The following procedure describes pouring a 36 cm Long Ranger gel Long Ranger Singel Packs 377 36 cm WTR product 50691 BioWhittaker Molecular Applications purchased from Intermountain Scientific using an Otter apparatus also available from Intermountain Scientific NOTE Orientation of the plate with respect to the gel must be consistent from gel to gel The product number and serial number etched onto each plate must be on the outside of the sandwich A For each gel place a clean notched glass plate with the inside face up on the Otter Wet two spacers and lay them on top of the plate flush with the outside edge of the plate Place a clean rabbit ear glass plat
77. to 6 with 10 NaOH Add 420 g sodium chloride NaCl and 55 2 sodium phosphate monobasic monohydrate NaH PO Adjust pH to 7 4 using 10 NaOH about 10 ml Bring up to 2 liters and mix Autoclave A NT Amount NaCl lot NaH PO H 0 lot Na EDTA 2H50 lot 10N NaOH lot pH Preparer 20XSSPE Page 227 of 255 Appendix T DNA Stock Solutions Log 20X SSPE Buffer Model CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 APPENDIX U DNA STOCK SOLUTIONS Loc 10X TBE 890MM TRIS 890MM Boric AciD 20MM EDTA 1L Solution Preparation To approximately 800 ml ultrapure gt add 40 ml 0 5M EDTA pH8 0 Add 108 Tris base and 55 g Boric acid to the diluted EDTA solution Stir until dissolved Adjust volume to 1L with ultrapure water Filter through a Nalgene 0 2 or 0 45 micron filter unit and store at room temperature in clear container Note If a white precipitate is noted in 10X TBE the buffer should be discarded and remade Date Prepared Amount 0 5 M EDTA lot Tris base lot Boric acid lot Preparer 10XTBE2 Page 228 of 255 Appendix U DNA Stock Solutions Log 10X TBE CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 APPENDIX V DNA STOC
78. to analyze the sample will store the blood stain card or buccal sample upon completion of analysis Page 179 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 This Page Intentionally Left Blank Page 180 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 APPENDIX Page 181 of 255 Appendix CBI DNA SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 This Page Intentionally Left Blank Page 182 of 255 CBI DNA Analysis SOP Vers 2 1 Codes HOMicide TP1 SB1 1 5 1 HR1 SEXual Assaults TP1 SB1 1 SP1 HR1 OTHer Crimes TP1 SB1 1 5 1 HR1 DataBaSe TP1 SB1 1 SP1 FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 APPENDIX A ANALYST ACTIVITY AA FORMS OTH NCD XCD ROU NSF DBS DOC PRO DYC Included suspect Excluded suspect Routine sample run no inclusion or exclusion Samples Not Sufficient for analysis Number of Examinations Number of Specimens Hours per SOP Included suspect Excluded suspect Routine sample run no inclusion or exclusion Samples Not Sufficient for analysis Number of Examinations Number of Specimens Hours per SOP Included suspect Excluded suspect Routine sample run no inclusion or exclusion Samples Not Sufficient
79. tooth utilizing sterile instruments Extirpate pulp tissue C Place the processed tooth tissue into a microcentrifuge tube If pulp tissue is limited in quantity a portion of the crushed dentate may be placed in the microcentrifuge tube To the sample add 300 ul organic stain extraction buffer 12 ul 1M DTT and 4 ul 10 mg ml Proteinase K solution Close the tube cap D Vortex for 1 second and spin in a microcentrifuge for 2 seconds to force the sample into the extraction fluid Incubate the tube at 56 C overnight F Spin in a microcentrifuge for 2 seconds to force condensation into the bottom of the tube G In a fume hood add 300 ul phenol chloroform isoamyl alcohol to the extract Vortex low speed the mixture briefly to attain a milky emulsion Spin the tube in a microcentrifuge for 3 minutes Note If necessary additional organic extractions may be performed prior to the purification steps H To a Microcon concentrator add 100 ul Transfer the aqueous phase from the tube in Step G to the concentrator Avoid pipetting organic solvent from the tube into the concentrator l Place a cap on the concentrator and spin in a microcentrifuge at 2500 x g for 10 minutes J Add 100 ul to the concentrator Replace the spin cap and spin the assembly in a microcentrifuge at 2500 x g for 10 minutes K Remove the cap and add a measured volume of that is between 20 ul and 200 ul to the concentrator Remove
80. with care The dots on the AmpliType PM DNA Probe Strip correspond to the following alleles The dot for each locus is positive in the presence of the A allele NOTE The A dot for the GYPA locus is positive in the presence of both the A allele and the A allele Both the GYPA AB and GYPA A and B variant alleles observed in lt 8 of the African American population may produce a slightly imbalanced heterozygous signal The dot for each locus is positive in the presence of the B allele The dot for the HBGG and GC loci is positive in the presence of the C allele For LDLR GYPA and D7S8 three genotypes are possible AA BB and For HBGG and GC six genotypes are possible AA BB CC AB AC and BC An example of a developed AmpliType PM DNA Probe Strip using PCR product amplified from 2 ng of Control DNA 1 is shown in Figure 2 A sample from a single individual will produce balanced dot intensities within each locus for which the individual is heterozygous Page 79 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 LDL GYPA HBGG D7S8 GC 0 0 Figure 2 AmpliType PM types for Control DNA 1 The AmpliType PM types for Control DNA 1 are LDLR BB GYPA AB HBGG AA D7S8 AB GC BB The dot intensities of the GYPA and D7S8 loci are balanced i e the intensities of the A and B dots within eac
81. 123 0 0131 34 2 0 0140 0 0128 0 0123 0 0131 35 0 0279 0 0128 0 0123 0 0131 35 2 0 0140 0 0128 0 0123 0 0131 36 0 0140 0 0128 0 0123 0 0131 gt 36 0 0140 0 0128 0 0123 0 0131 Page 201 of 255 Appendix D Allele Frequencies CBI DNA Analysis SOP Vers 2 1 06 13 02 4 43 PM CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 ALLELE FREQUENCIES CONTINUED 018551 Allele 2 acd 2 mE N 180 N 196 N 203 N 191 lt 11 0 0139 0 0128 0 0123 0 0131 11 0 0139 0 0128 0 0123 0 0157 12 0 0583 0 1276 0 1059 0 1361 13 0 0556 0 1225 0 1700 0 1178 13 2 0 0139 0 0128 0 0123 0 0131 14 0 0639 0 1735 0 1700 0 1309 14 2 0 0139 0 0128 0 0123 0 0131 15 0 1667 0 1276 0 1379 0 1911 15 2 0 0139 0 0128 0 0123 0 0131 16 0 1889 0 1071 0 1158 0 1414 17 0 1639 0 1556 0 1379 0 1073 18 0 1306 0 0918 0 0517 0 0550 19 0 0778 0 0357 0 0370 0 0471 20 0 0556 0 0255 0 0172 0 0288 21 0 0139 0 0128 0 0197 0 0131 212 0 0139 0 0128 0 0123 0 0131 2 0 0139 0 0128 0 0123 0 0131 gt 22 0 0139 0 0128 0 0123 0 0131 202 of 255 Appendix Allele Frequencies CBI DNA Analysis SOP Vers 2 1 06 13 02 4 43 PM CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 ALLELE FREQUENCIES CONTINUED 055818 African i Southwest Southeast Allele American caucasian Hispanic Hispanic
82. 2 could not be excluded Therefore the RPNE is going to equal the frequency of a 15 15 and 15 X or RPNE locus 2 p2 2p 1 p 1 1 2 equation 2 The probability of exclusion PE would then become PE iocus 1 1 RPNE iocus 1 PE iocus 2 1 RPNE ocus 2 The combined PE for all loci tested would be Page 119 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 PE combined 1 RPNE locus 1 RPNE locus 2 _ RPNE locus n The proportion of the population excluded as the possible parent s is expressed in report form as 100 PE combinea in terms of a percent For any case in which the parents of a living or dead person are unknown referred to as reverse parentage the equations above do not change Examples of cases involving reverse parentage are missing person cases involving suspected crime scenes a single bone found in a particular location or an abandoned child There will be times in working criminal parentage cases that one of the biological parents will be known i e a sexual assault in which a baby is conceived In this case the mother of the child would be known and it may be possible to determine which obligate allele she passed to her child In cases such as these it may be possible to determine specifically which obligate allele must have been passed from the biological father Mother 8 9 Child 8 14 Obligate allele 14 OR Mother 8 9 Child 8 Obligate allele 8
83. 2 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 CODIS AND CODIS SEARCHES CODIS Combined DNA Index System is a database that was developed by the FBI to track the results of the DNA analyses performed on forensic specimens and convicted offenders Accessibility to CODIS is limited to NDIS National DNA Index System qualified personnel CODIS security is maintained by keeping access to the server limited to CODIS Administrators The server is located in a secure locked room Access to CODIS software programs and data is limited to NDIS qualified personnel and is password protected Specimen identification is coded prior to entry into any CODIS database Page 23 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 This Page Intentionally Left Blank Page 24 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 DNA PREPARATION AND STORAGE OF SAMPLES FOR CONVICTED OFFENDER DATABASE SAMPLE SUBMISSION REQUIREMENTS Requirements Samples A Blood samples must consist of at least one Lavender purple top Vacutainer EDTA tube from each offender B Buccal samples must be dried on the appropriate substrate and then sealed in a protective package Labeling and Packaging A The blood tube will be labeled with the offender s name first and last submitting ag
84. 2002 Clean disposable gloves should be worn throughout the DNA Hybridization and Color Development steps to avoid soiling the DNA probe strips and to protect fingers from the 95 C block Gloves and lab coats should be discarded when leaving the work area to avoid transport of amplified DNA from the work area A Heat the rotating water bath to 55 C and maintain the temperature between 54 C and 56 C It is essential to check the temperature with a calibrated complete immersion thermometer before the hybridization step is performed B The water level should be inch above the shaker platform The water level should not be higher than 74 inch since higher levels may result in water splashing into the wells of the tray An empty tray can be used to test the water level prior to use A rotating water bath set at 50 to 90 rpm is necessary for the hybridization and wash steps Maintain the rotation and the temperature of the water bath throughout Section Do NOT use a hot air shaker C Heat an incubator to 50 to 55 C and warm the Hybridization Solution and the PM DQA1 Wash Solution to 50 to 55 C All solids must be completely dissolved all solutions should be well mixed before use D Using filter forceps remove the required number of AmpliType DNA Probe Strips from the tube Using the pen included with the AmpliType DNA Typing Trays label each strip in the space at the right edge of the strip The use of other pens is no
85. 2002 Table 4 ALLELES POSITIVE SYSTEM LOCUS EU PRESENT IN COLOR CONTROL LADDER 9947A Both D3S1358 111 140 12 19 5 14 15 Blue Profiler Plus VWA 154 195 11 21 Seis 17 18 Profiler Plus FGA 216 265 18 26 26 2 27 30 jus 23 24 Both Amelogenin 103 109 ec Profiler Plus 0851179 124 171 8 19 JOE 13 13 Green 24 2 25 28 28 2 29 29 2 30 30 2 joe Profiler Plus 021511 187 240 31 31 2 32 322 Goo 39 30 33 33 2 34 34 2 35 35 2 36 38 9 10 10 2 11 13 xx Profiler Plus 018851 270 342 13 2 14 14 2 15 15 19 26 Profiler Plus 059818 131 170 7 16 NED 14 11 Yellow Profiler Plus 0138317 205 233 8 15 NED 11 11 Yellow Both 075280 256 293 6 15 NED 10 11 Yellow 1 samples Page 95 of 255 CBI DNA Analysis SOP Vers 2 1 The allelic ladders provided in the kit are used to determine the genotypes of the CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 Table 4 ALLELES POSITIVE SIZE RANGE pRESENT IN CONTROL SYSTEM LOCUS bp LADDER 9947A Cofiler THO1 167 187 5 9 9 3 10 8 9 3 Cofiler TPOX 215 243 6 13 8 8 Cofiler CSF1P0 279 318 6 15 Cofiler D16S539 229 270 5 8 15 Sizes are actual base pair size of alleles in the Profiler Plus and Cofiler ladders including the nucleotide addition The sizes obtained in Genotyper may vary from the actual fragment sizes due to electrophoretic effe
86. 30 room temperature reagent grade 100 ethanol to the bottle Do NOT use ethanol that has been stored in a metal container Do not use 95 ethanol or other alcohols Recap the bottle Seal the stopper with Parafilm amp Shake in an upright position on an orbital shaker for 2 hours or until completely dissolved Store bottle at 2 to 8 C Under these conditions the Chromogen Solution is stable for six months after preparation B AmpliType PM DQA1 PCR Reaction Mix Upon first use of the AmpliType PM DQA1 PCR Amplification and Typing Kit remove the bottle of AmpliType PM DQA1 PCR Reaction Mix and carefully aliquot 40 ul into autoclaved tubes GeneAmp Autoclave Thin Walled Reaction Tubes for the DNA Thermal Cycler 480 using a dedicated positive displacement repipettor or a pipettor with hydrophobic filter plugged tips CAUTION This step must be performed either in a biological hood or ina room free from amplified DNA Page 68 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 Pipet 2 ul of Bovine Serum Albumin Fraction 5 8 ug ul into each tube using sterile pipette Insure that the tubes are capped tightly Place PCR Reaction Mix tubes in a rack not used for DNA preparation or amplified DNA handling Store tubes separated from any source of DNA at 2 to 8 C NOTE All of the AmpliType PM DQA1 PCR Reaction Mix should be aliquoted at the same time to
87. 340 bp in size The ladder used by Genotyper to call the alleles of the samples analyzed must be correctly labeled with the correct allele designations Genotypes are determined from the diagnostic peaks of the appropriate color and size range for a particular locus The minimum peaks threshold must be 150 RFU or greater to be considered for match purposes Peaks between 50 and 150 RFU will be interpreted with caution and utilized for the following A If it provides exculpatory information it can be noted on the report B If it indicates the presence of a mixture it can be noted in the report Homozygous allele peak heights are in general approximately twice that of heterozygous peaks as a result of doubling of the signal from two alleles of the same size Caution should be used with the interpretation of low RFU single allele peaks They may indicate homozygous alleles but could also be the result of preferential amplification Genotypes can still be interpreted even when some loci amplify poorly Each locus is independent of the others allowing for results to be interpreted and a genotype profile obtained at the other loci Poor amplification at a locus may be due to degraded DNA presence of inhibitors extremely low or excessive input DNA or primer mismatch Page 111 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 Samples may not be re injected for longer than 5 seconds
88. 5 5 uL of primer set The above formulation provides a slight overfill to allow for volume lost in pipetting Be sure to include enough Master Mix for a positive amplification control a reagent blank and a negative amplification blank Mix thoroughly by vortexing at medium speed for 5 seconds Spin the tube briefly in a microcentrifuge to remove any liquid from the cap Sample preparation is detailed below Final amplification volume is 25 uL SAMPLE PREPARATION CHELEX SAMPLES USING COFILER AND PROFILER PLUS KITS A B G H Prepare a DNA TE dilution of each sample The final amount of input DNA is 0 5 to 2 ng diluted in TE to a final volume of 20uL Determine the number of samples to be amplified Place the required number of amplification reaction tubes into a rack including tubes for the positive control reagent blank and negative amplification blank Treat the tubes with ultraviolet light for at least 20 minutes Prepare Master Mix according to Formulation instructions Dispense 30 ul of Master Mix into each reaction tube If using the 480 Thermal Cycler add one drop of mineral oil supplied with kit to each tube Open each sample tube add 20 ul of the appropriate DNA TE sample preparation and then close the tube Make sure to have only one tube open at a time Do not mix or vortex the tube Add 20 yl of the reagent blank to the tube for the reagent blank control Add 20 yl of the control DNA provided
89. 8 AMPLIFICATION REAGENTS ss roten taa hem utt rate oec tsm o e p m ne e tuta 98 QUANTIFICATION PROCEDURE 8 4 4408 98 TZAOMPEIRICATIONGSe ea A tt t C dd o d dat 99 SAMPLE PREPARATION S2 dd ni Bird d o cd ae a PLU AO n a Pc d 99 AMPLIFICATION MIX 100 STR TYPING BY CAPILLARY GEL ELECTROPHORESIS 101 SETTING UP THE INSTRUMENT a e Cot cud cues o cont aah cas cds Gaus cud 101 PREPARING FOR A RUN inate phatase 106 GENESCAN GENOTYPER 109 INTERPRETATION OF DATA 110 EVALUATION OF STR DATA 111 pate Mona ite daviedeie tates tase sate Mote ce dae dat ote up on 112 NON TEMPLATE NUCLEOTIDE ADDITION 113 lI 114 SPIKES AND OTHER ANOMALIES s asc toe 114 OFF SCALE DATA MT 114 OPFESLADDER ALLELES reed iru ai otia tel 114 a a 115 NEGATIVE AMPLIFICATION 4 8444 115 POSITIVE EXTRACTION AND AMPLIFICATION CONT
90. 9 N 240 gt 6 0 0119 0 0123 0 0120 0 0104 6 0 0119 0 0123 0 0120 0 0104 7 0 0119 0 0172 0 0215 0 0104 8 0 1738 0 1626 0 0981 0 1417 9 0 1571 0 1478 0 0479 0 1250 10 0 3238 0 2906 0 3062 0 2667 11 1 0 0119 0 0123 0 0120 0 0104 11 0 2238 0 2020 0 2895 0 2271 11 3 0 0119 0 0123 0 0120 0 0104 12 0 0905 0 1404 0 1914 0 1875 13 0 0191 0 0296 0 0383 0 0354 14 0 0119 0 0123 0 0120 0 0104 gt 14 0 0119 0 0123 0 0120 0 0104 Page 205 of 255 Appendix D Allele Frequencies CBI DNA Analysis SOP Vers 2 1 06 13 02 4 43 PM CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 ALLELE FREQUENCIES CONTINUED CSF1PO African i Southwest Southeast Allele American caucasian Hispanic Hispanic mE N 210 N 203 N 209 N 240 gt 6 0 0119 0 0123 0 0120 0 0104 6 0 0119 0 0123 0 0120 0 0104 7 0 0429 0 0123 0 0120 0 0104 8 0 0857 0 0123 0 0120 0 0104 9 0 0333 0 0197 0 0120 0 0125 10 0 2714 0 2537 0 2536 0 2542 10 3 0 0119 0 0123 0 0120 0 0104 11 0 2048 0 3005 0 2656 0 2958 12 0 3000 0 3251 0 3923 0 3563 12 1 0 0119 0 0123 0 0120 0 0104 13 0 0548 0 0714 0 0646 0 0688 14 0 0119 0 0148 0 0120 0 0104 15 0 0119 0 0123 0 0120 0 0104 gt 15 0 0119 0 0123 0 0120 0 0104 Page 206 of 255 Appendix D Allele Frequencies CBI DNA Analysis SOP Vers 2 1 06 13 02 4 43 PM CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 ALLELE FREQUENCIES CONTINUED
91. 9 and the 36 allele of D21S11 ladder is 236 32 bp then the peak will be designated 021511 36 2 Alleles smaller than the ladders lowest molecular weight allele i e A will be designated lt A Alleles greater than the ladder s largest molecular weight allele i e B will be designated gt Significant room temperature fluctuations may result in size variation between injections during a single run such that allelic ladder peaks differ by more than 0 5 bp from allelic peaks in other injections Genotyping with another allelic ladder may alleviate this problem If not then the cases may be broken down into sections such that a ladder associated with that case can be used to correctly type the samples associated with that case REAGENT BLANKS A reagent blank tests for possible presence of contamination of the extraction reagents and or supplies by an adventitious source of DNA The adventitious source of DNA may be non amplified DNA or PCR product Peaks gt 50 RFU not attributable to artifacts located between 100 and 350 bp or at 75 bp and 400 bp indicate the presence of contamination and none of the samples extracted or amplified with the reagent blank will be considered inclusive for match purposes If they cannot be re amplified then these samples can be considered for exclusion purposes If artifactual peaks occur then the reagent blank must be re injected NEGATIVE AMPLIFICATION BLANK A negative amplification blank is a test fo
92. A ANALYSIS SOP VERSION 2 1 JUNE 2002 APPENDIX AO DNA WORKING SOLUTIONS LOG TE BUFFER MODEL 10MM TRIS HCL 0 1MM EDTA 8 0 1L Solution Preparation Mix together 10 ml 1M Tris HCl pH 8 0 0 2 ml 0 5M EDTA and 990 ml ultrapure water and sterilize by autoclaving Store at room temperature Date Amount Tris HCl 0 5M EDTA Preparer Verified by Prepared pH 8 0 lot lot initials date TEBUFF Page 248 of 255 Appendix AO DNA Working Solutions Log TE Buffer CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 APPENDIX AP DNA WORKING SOLUTIONS Loc TRIS EDTA NACL MODEL 10MM TRIS HCL 100MM NACL 1MM EDTA PH 8 0 100 ML Solution Preparation Add 1ml 1M Tris HCl to approximately 75 ml ultrapure water To this solution add 0 584 g NaCl and 200 ul 0 5M EDTA Stir until dissolved Adjust the pH to 8 0 with 1N NaOH and bring to a final volume of 100 ml with ultrapure water Autoclave and store at room temperature Date 1M Tris 0 5 M EDTA 1N NaOH Amount NaCl lot pH Preparer Verified date Prepared pH 8 0 lot lot lot TRIS ETDA NACL Page 249 of 255 Appendix AP DNA Working Solutions Log Tris EDTA NaCl CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002
93. ALYSIS SOP VERSION 2 1 JUNE 2002 In a case where a small isolated population e g Native American is relevant frequency calculations for that population will use 0 03 for 0 Note Amelogenin is not incorporated in the population statistics Popstats in the FBI CODIS system may be utilized to obtain the statistical analysis results associated with a given DNA profile Minimum allele frequencies A minimum allele frequency estimation is calculated for STR loci which demonstrate alleles with a low frequency of occurrence The intent of this application is to set a lower limit for the frequency for such rare alleles and consequently produce a conservative allele frequency estimate that does not underestimate the allele s frequency of occurrence The approach utilized is a basic procedure described previously by Budowle et al 1991 and National Research Council 1996 The minimum allele frequency is calculated using the following expression I 2n where n represents the sample size individuals Pyan Mixture calculations A mixture calculation may be performed using the allele frequencies of each allele represented in a profile with intensity greater than or equal to 50 RFU to determine the probability of selecting an unrelated individual in the population who could be a potential contributor to the mixture To calculate the frequency of occurrence for individual STR loci and multi locus STR DNA profiles when a mixture is pr
94. ANALYSIS SOP VERSION 2 1 JUNE 2002 Reading and interpreting the AmpliType PM DQA1 DNA Probe Strips The AmpliType PM DNA Probe Strips have been spotted with a total of fourteen sequence specific oligonucleotide probes to distinguish the alleles of five genetic loci a mixture of two probes is spotted at the GYPA A allele position Under the AmpliType hybridization conditions the typing probes will bind specifically to the PCR product containing the alleles designated on the AmpliType PM DNA probe strip To read the developed AmpliType PM DNA Probe Strip the 5 dot is examined first and then each locus is examined separately The standard probe S on the AmpliType PM DNA Probe Strip is identical in sequence to the control probe C on the AmpliType HLA DQA1 DNA Probe Strip and detects all of the HLA DQA1 alleles The S dot is designed to be the lightest typing dot on the PM DNA Probe Strip and acts as a minimum dot intensity control for the remaining probes A DNA probe strip with no visible S dot shall not be typed for any locus When an S dot is visible on the AmpliType PM DNA Probe Strip the intensities of the dots at the remaining 12 positions are compared to the intensity of the 5 dot Those dots that appear either darker than or equivalent to the S dot are considered positive Each positive dot indicates the presence of the corresponding allele Dots that are lighter than the S dot should be interpreted
95. APPENDIX G AMPLIFICATION DATA MODEL Extraction date Analyst QB PM DQA1 LOT LOT 4 Case Item CONTROL DATE CONTROL DATE Date Ng ul ul DNA WELL Page 212 of 255 Appendix G Amplification Data Model CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 APPENDIX H FREQUENCIES MODEL Case Number Date Analyst Southwestern African American Caucasian Hispanics DQA1 LDLR GYPA HBGG 0758 GC D1S80 TOTAL Page 213 of 255 Appendix H Frequencies CBI DNA Analysis SOP Vers 2 1 Analyst Case Item CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 APPENDIX STR DILUTION AND AMPLIFICATION WORKSHEET MODEL QuantiBlot Lot Exp Date ng ul Profiler Plus Lot Exp Therm Date Dilution Lot Exp Therm Date TE Lot Date DNA ul ul Cofiler Well CO N O On RO gt Page 214 of 255 Appendix STR Dilution and Amplification Worksheet Model CBI DNA Analysis SOP Vers 2 1 TAQ PRIMERS 11
96. ASH SOLUTION MODEL d 241 DNA WORKING SOLUTIONS LOG PROTEINASE 10 MG ML ean rate tee DEP oui M Su 242 DNA WORKING SOLUTIONS LOG QUANTIBLOT PREWETTING SOLUTION MODEL 243 DNA WORKING SOLUTIONS LOG QUANTIBLOT SPOTTING SOLUTION 244 DNA WORKING SOLUTIONS LOG WASH SOLUTION MODEL tc dada 245 DNA WORKING SOLUTIONS LOG QUANTIBLOT PM DQA1 HYBRIDIZATION SOLUTION 246 DNA WORKING SOLUTIONS LOG 20 SARKOSYL MODEL 247 DNA WORKING SOLUTIONS LOG TE BUFFER MODEL 248 DNA WORKING SOLUTIONS LOG TRIS EDTA NACL MODEL 249 DNA CHEMICAL LOG MODEL o ED RESO 250 DNA QA QC FORM 251 Page 9 of 255 Table of Contents Continued CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 EXTRACTION SUMMARY SHEET MODEL 252 CBI DNA DATABASE SAMPLE EXTRACTION AMPLIFICATION WORKSHEET MODEL ideo sea 253 GENESCAN GENOTYPER REVIEW SHEET 0 254 3200 LOAD SHEET MODEL te mede donus 255 Page 10 of 255 Table of Contents Continued CBI DNA Analysis SOP Vers 2 1 CBI FORENSI
97. ATING BLOCKS 19 obe unitate eee ee 19 CENTRIFUGES stottescist esee emt p esse bot pese pent esee pese test EE 19 f uie uti 19 AA ING ts fede ass ae 19 WATER PURIFICATION SYSTEMS 20 REFRIGERATORS FREEZERS 20 C AND 70 C AND INCUBATORS eade dee e ied 20 ABI 377 ABI 310 AND ABI sce eost eee eot 20 MATERIAL SAFETY DATA SHEETS 20 1 of 255 Table of Contents CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 LOGBOOKS FOR CHEMICALS COMMERCIAL KITS IN HOUSE REAGENTS AND CRITICAL REAGENTS 20 RECORDS OF EQUIPMENT TEMPERATURES MAINTENANCE AND GAELIBRATIONS C a te teen dones 20 PROFIGIENGY TESTIN G satio octaua ecouter tees 21 TESTING REQUIREMENT PRIOR TO 21 PERIODIC TESTING REQUIREMENT ie 21 TESTING REQUIREMENT PRIOR TO DATABASE ANALYSIS 22 PERIODIC TESTING REQUIREMENT 552 2656 Fre enr ero dee eet ep tete 22 CODIS AND CODIS S
98. Analysis range incorporate 75 to 400 base pair range 2 Data processing check baseline multi component and light smoothing 3 Peak detection B50 G50 50 and R150 RFUs The RFU may be increased to reduce signal to noise ratios 4 Size call range min 75 max 400 5 Size calling local southern method 6 Split peak correction none Verify all peaks in the GS500 size standard have been correctly assigned for each sample and that the 250 peaks throughout the analysis sample set are within one base pair Examine reagent blanks and negative amplification blanks for anomalous peaks Examine each sample making note of any anomalies pull up spikes A peaks dye peaks peak height variation etc or other electrophoretic aberrations noisy baseline matrix quality incomplete electrophoresis etc Examine and check to make sure all ladder alleles are labeled with the correct designation and that a correct ladder is applied to each analysis sample set Check and confirm off ladder alleles making note of any samples needing re analysis or re run Check and insure that each sample is correctly labeled with allele designation and peak height Print electropherograms of all samples controls and ladders used in analysis Save file according to conventions INTERPRETATION OF DATA The interpretation of casework results is a matter of both professional judgement and expertise The following objective criteria are to be used
99. BLOOD OR BLOODS TAINS ete oen eee cete cece 41 VAGINAL SWABS OR SEMEN STAINS 42 Page 2 of 255 Table of Contents Continued CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 SALIVA STAINS 44 ENVELOPE FLAPS ORAGTAMBOS 45 CIGARETTE BUTTS dite reo oret e e 46 UM EM 47 HAIRS 48 UNMOUNTED HAIR SPECIMENS 2 rae te one 48 SLIDE MOUNTED HAIR SPECIMENS cuddeuhdceddeubecnbdevSdced subdad eubedene 49 coena oe Rp M D LL 49 TEETH too teu DLE 51 BLOOD EXTRACTION USING FTA 5 54 QUANT BLO T 56 PERFORMANCE CHARAGCTERISTIGQS desk ds 62 TROUBLESHOOTING cae ade DIL eO o e DI teo o e cic asc 62 AMPLITYPE PM EDO teet meet tet eset ei beo Ree 68 STORAGE AND STABILITY 2 22 22 Seed Ind Sach 68 PREPARATION OF REAGENTS 68 PISECADTIONS 5 reset
100. C LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 DNA LABORATORY ORGANIZATION The Colorado Bureau of Investigation DNA Laboratory is organized according to the most recent guidelines established by the SWGDAM Scientific Working Group on DNA Analysis Methods Because of the sensitivity of PCR based DNA analyses and the consequent potential for sample contamination precautions must be implemented at each stage of the analytical process to avoid concurrent and future contamination These sources as well as the appropriate measures to prevent such contamination follow Sample contamination with human genomic DNA environment surrounding a sample This chance contamination could be introduced with the analyst s own DNA during handling or by DNA from other specimens examined before the sample in question Contamination between samples during sample preparation from pipette tips tools aerosols This is a specific type of environmental contamination and is of concern particularly when samples of high DNA concentration are prepared simultaneously with low level DNA samples Contamination of a sample with amplified from a previous PCR reaction referred to as PCR product carryover Since the number of copies of amplified DNA in a completed PCR reaction is so high the inadvertent transfer of even a minute volume to a sample yet to be amplified may result in the amplification and typing of the contaminatin
101. CATION PROCEDURE For samples extracted with Chelex an estimate of the DNA concentration of the samples will be conducted prior to STR analysis CBI uses the QuantiBlot biotin streptavidin HRP method to evaluate the DNA concentration of each extracted sample In general 0 75 to 2 0 ng of DNA is optimal for STR amplification although results may be obtained outside of this range See the QUANTIBLOT section of this SOP for more details The FTA paper extraction procedure does not require quantification AMPLIFICATION All PCR setup steps must be performed in the DNA extraction PCR setup room of the laboratory using hoods reagents and pipettors dedicated to this area The reagents required for the amplification of the thirteen core STR loci are included in two kits the AmpF STR Profiler Plus PCR Amplification Kit and the AmpF STR Cofiler PCR Amplification Kit or in a single combined kit the AmpF STR Identifiler Amplification Kit For each amplification combine DNA template PCR reaction mix Primer set Profiler Plus Cofiler Identifiler and DNA polymerase AmpliTaq Gold in a reaction tube and subject this mixture to a series of controlled temperature changes in the thermal cycler The AmpF STR Identifiler Amplification Kit contains primers for two additional loci 0251338 0195433 THERMAL CYCLER PARAMETERS The following parameters are used to amplify the AmpF STR Profiler Plus Cofiler and Identifile
102. CAUSE Hydrogen peroxide was not added or too much was added to the Color Development Solution Hydrogen peroxide inac tive Chromogen TMB was not added to the Color Development Solution The original AmpliType HLA DQa_ instead of AmpliType PM and PM DQA1 typing protocol was followed Quantity of DNA test sample is below the assay sensitivity Excess amounts of Enzyme Conjugate HRP SA added to Enzyme Conjugate Solution Exposure to strong light and oxidizing agents Page 84 of 255 CBI DNA Analysis SOP Vers 2 1 RECOMMENDED ACTION Make new Color Develop ment Solution using 3 hydrogen peroxide repeat test Make new Color Develop ment Solution using new bottle or dilution of hydro gen peroxide repeat test Make new Color Development Solution add ing Chromogen TMB re peat test Repeat test following protocol in Section DNA Hybridization Repeat amplification in creasing DNA quantity to 22 ng Dilute new Enzyme Conjugate Solution with correct amount of Enzyme Conjugate HRP SA Cover tray with foil during Color Development steps Repeat test using deion ized or glass distilled water for water rinses Store strips in the dark away from oxidizing agents CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 OBSERVATION 5 Presence of unex pected or additional alleles in the amplified Control DNA 1 sample 6 Signals weaker than S or C d
103. CB Forensic Laboratory Biological Science Training Plan each analyst will be required to successfully complete DNA analysis on a minimum of 40 blood samples and a range of samples typically encountered in forensic casework prior to conducting independent DNA casework In addition each analyst will be required to successfully complete one proficiency qualifying test PERIODIC TESTING REQUIREMENT Each analyst who conducts DNA testing on casework specimens will be required to complete a minimum of one external proficiency test every 183 days to demonstrate the reliability of the Laboratory s analytical methods as well as the interpretive capability of the analyst Page 21 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 TESTING REQUIREMENT PRIOR TO DATABASE ANALYSIS Following the CB Forensic Laboratory Biological Science Training Plan each analyst will be required to successfully complete DNA analysis on a minimum of 40 blood samples prior to conducting independent DNA database analyses In addition each analyst will be required to successfully complete one proficiency qualifying test PERIODIC TESTING REQUIREMENT Each analyst who conducts DNA testing on database specimens will be required to complete one external proficiency test every 183 days to demonstrate the reliability of the Laboratory s analytical methods as well as the interpretive capability of the analyst Page 2
104. CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 TABLE OF CONTENTS TOPIC PAGE DNA LABORATORY ORGANIZATION cccccccccecececeeececceceeeeceeeceseccceseseseeeeseeeess 11 EVIDENCE EXAMINATION WORK AREA 12 DNA EXERAGIAONSAES E 5 dc esate coco eel enol 12 PCR SE LUP AREA des 13 PCR AMPLIFICATION AND TYPING AREA 13 DORN ne nd RO td GT RPE daten RA dores dee ee 14 CLEANING AND 14 EVIDENCE HANDUN 15 PREPARATION OF SAMPLES FOR DNA 15 15 PRESERVATION d 15 MISCEEPANEDOUS EVIDENGCE enitn tes 16 MINIMAL EVIDENCE SUBMITTED FOR DNA ANALYSIS 16 QUALITY CONTROL MEASURES tod ed dod o dod ee inet 17 REAGENTS cc caters cok eed cat ck one none E nnne ass 17 CRITICAL REAGENTS dnc 17 STANDARDS AND GONT 5 ueceeuceceas 17 EQUIPMENT CALIBRATION AND MAINTENANCE 19 STAR WAY GER eo XS 19 HYBRIDIZATION BATHS AND HE
105. CCAL SWAB STANDARDS STAMPS ENVELOPE FLAPS CIGARETTE BUTTS A Cut a sample of the item to be analyzed and place into a sterile 1 5 ml microcentrifuge tube Use a clean cutting surface for each different sample Pipet 1ml of TE Buffer into the sterile 1 5 ml microcentrifuge tube Incubate at room temperature for 15 to 30 minutes Mix occasionally by inversion or gentle vortexing Spin in a microcentrifuge for 2 to 3 minutes at 10 000 to 15 000 x g Carefully remove supernatant all but 20 to 30 and discard Leave the fabric substrate in the tube with the pellet Page 37 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 Add 5 Chelex to a final volume of 200 ul F Incubate at 56 C for 15 to 30 minutes or overnight Overnight is optimal Vortex at high speed for 5 to 10 seconds Spin in a microcentrifuge for 5 to 10 seconds at 10 000 to 15 000 x g H Incubate in a boiling water bath for 8 minutes l Vortex at high speed for 5 to 10 seconds J Spin in a microcentrifuge for 2 to 3 minutes at 10 000 to 15 000 x g K The sample is now ready for the PCR Amplification process L Store the remainder of the supernatant at 2 to 8 C or frozen To re use repeat Steps I through TISSUES Tissues should be stored at 20 C until used A Place a portion of the tissue on a clean surface Remove a piece of tissue approximately 1 and tease apart or cut into
106. DIS database Page 170 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 2 Analysts may request a remote search of the SDIS database The profile is entered into LDAS it is not marked for transfer to LDIS The remote search request is then sent to SDIS and executed by the State Administrator B In general ALL samples once entered into LDIS will be marked for SDIS C An SDIS upload from each local laboratory may occur weekly in conjunction with the State system upload to NDIS The Local CODIS Administrator must conduct the SDIS upload D Routine SDIS autosearches will be performed COMMUNICATION PROTOCOL FOR A POSSIBLE CODIS HIT NOTE The AIC and the Deputy Director of CBI will be notified of CODIS hits resulting in investigations aided NOTE Release of personal information by the CBI is in accordance with the following federal legislation The DNA Identification Act of 1994 and the Privacy Act Additionally C R S 24 72 305 which governs public inspection of criminal justice records denies public access to the results of DNA testing pursuant to state statutes regarding Convicted Offender samples A Upon conducting a search of the LDIS index a possible hit match is made The following course of action shall be taken 1 copy of the match report will be printed This report will give the case designation for the case to which the possible match was made
107. DQA1 PCR Amplification and Typing Kits are developed and manufactured by Roche Molecular Systems Inc RMS Each lot of the AmpliType PM and AmpliType PM DQA1 PCR Amplification and Typing Kits is carefully tested by RMS to insure that the kits perform according to specifications and are free from interfering contaminants The user of the AmpliType PCR Amplification and Typing Kits will be able to amplify and type a minimum of 2 ng of Control DNA 1 when employing the protocols and reagents provided in the kits In the laboratories of RMS the kit components have been used successfully to type samples containing less than one ng of human DNA TROUBLESHOOTING OBSERVATION POSSIBLE CAUSE RECOMMENDED ACTION 1 No signal or faint No PCR amplification or Repeat test from the signal from both the Control DNA 1 and the DNA test samples at all loci insufficient PCR amplifi cation of all markers Improper hybridization or assay condition No added or insufficient DNA added to PCR Reaction Mix AmpliType PM Primer Set not added to PCR Reaction Mix Page 82 of 255 CBI DNA Analysis SOP Vers 2 1 amplification step Repeat test from hybridi zation step Add gt 2 ng DNA repeat test from amplification step Add AmpliType PM Primer Set repeat test from amplification step OBSERVATION CBI FORENSIC LABORATORY JUNE 2002 POSSIBLE CAUSE GeneAmp PCR Instru ment System failure or wrong program
108. E 2002 Table 5 POSITIVE SIZE RANGE ALLELES PRESENTIN DYE SYSTEM LOCUS pude COLOR CONTROL 9947A Both 0381358 114 143 12 19 14 15 Profiler Plus WA 157 197 11 21 ce 17 18 Profiler Plus FGA 220 269 18 26 26 2 27 30 Pis 23 24 Both Amelogenin 106 112 X Y E Profiler Plus 0851179 127 172 8 19 JOE 13 13 Green 24 2 25 28 28 2 29 29 2 30 30 2 31 31 2 JOE Profiler Plus 021811 189 244 30 30 34 2 35 35 2 36 38 9 10 10 2 11 13 132 Profiler Plus D18S51 274 343 Jur em anc 15 19 Profiler Plus D5S818 134 171 7 16 NED 11 11 Yellow Profiler Plus 0138317 207 236 8 15 NED 11 11 Yellow Both 075280 259 294 6 15 NED 10 11 Yellow Cofiler THO1 169 190 5 9 9 3 10 8 93 Cofiler TPOX 218 247 6 13 JOE 8 8 Green Cofiler 281 317 6 15 JOE 10 12 Green Cofiler 0165539 233 273 5 8 15 fee 11 12 Sizes are actual base pair size of alleles in the Profiler Plus and Cofiler ladders including the nucleotide addition The sizes obtained in Genotyper may vary from the actual fragment sizes due to electrophoretic effects The allelic ladders provided in the kit are used to determine the genotypes of the samples Page 125 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 Table 6 SIZE DYE POSITIVE SYSTEM LOCUS RANGE IN COLO CONTROL
109. E EXTRACTION AMPLIFICATION WORKSHEET MODEL Identifiler Stain Cards Spotted INT Samples Cut INT Extraction Method CHELEX or FTA DATE INT LOT TE WASH CHELEX Detection Date Instrument Expiration date Thermocycler used 6 AA s Submitted 7 CODIS ENTRY Sample Info ul DNA Well PCR CBI LAST NAME OCA 1 2 total 3 of 4 samples 5 6 7 8 RXN MIX 9 19 X 10 5 ul 11 12 13 14 15 TAQ 16 17 X 0 5 ul 18 19 20 21 22 23 Primers 24 28 X 5 5ul 26 Page 253 of 255 Appendix AT CBI DNA Database Sample Extraction Amplification Worksheet Model CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 APPENDIX AU GENESCAN GENOTYPER REVIEW SHEET MODEL Project Runs Size Std Analysis Param Reviewed By ull Up OLA Spike I Up OLA Spike I Up OLA Spike I Up OLA Spike I Up OLA Spike OLA Spike I Up OLA Spike I Up OLA Spike OLA off ladder allele G Green B Blue R Red Y Yellow O Orange pk peak pks peaks bp basepair sz size std standard Param parameters height GS Genescan GT Genotyper ID Identifiler CO Cofiler PP ProfilerPlus A A addition incomplete D3 D381358 05 058818 CSF CSF1PO 07 075820 THO THO1 013 0135317 D16 D16S539 018 018551 021 021511 AMEL
110. EARGPIES 23 25 PREPARATION AND STORAGE OF SAMPLES FOR CONVICTED OFFENDER DATABASE iio eae 25 SAMPLE SUBMISSION REQUIREMENTS nennen nnn nnns 25 CBI HANDLING OF DATABASE SAMPLES 8 25 PREPARATION OF REAGENTS a citet ean etate ant 26 INORGANIC EXTRACTION 33 teles toa cS AEN MI I LN LM I ICI ae 33 PROCEDURE ae bed ale alae ia MM MM 33 SPERMATOZOA EXTRACTION IN THE PRESENCE OF CONTAMINATING CELLS POST 34 ducet ah vec conc tete octets 36 WHOLE SEMEN ni oio doar a dal ote 36 SEMEN STAINS es ceat bl idet bled able 36 ORAL SWABS WHICH CONTAIN SPERMATOZOA 36 BUG OAL SCRAPING ese 37 BUCCAL SWAB STANDARDS STAMPS ENVELOPE FLAPS CIGARETTE BUT TS ettet ics 37 TISSUES hee does 38 HAIR Mc 39 CONCENTRATION etate eterne 39 ORGANIC EXTRACTION 2 cccccccccccessssseeeeceeccscceasseneaeecceeescccaessseaeeeeeecssceeasens 41 WHOLE
111. ITION A ADDITION AmpliTaq Gold amp DNA Polymerase adds an additional nucleotide to the 3 ends of double stranded PCR product This addition creates a species n 1 nucleotide in length The n 1 species is considered an allele In some instances most notably in situations of sample overload and possibly suboptimal PCR conditions there can be incomplete addition n and two species will result that differ by one base pair n and n 1 The following considerations should be made to determine if a peak in the n position is an allele Determine if all the alleles are accounted for e Determine at which loci the n peaks are present 0351358 and THO1 seem to present this situation most readily Determine if common alleles are known to exist at this locus in the n position Page 153 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 Samples displaying split peak characteristics which indicates a high level of incomplete A addition may be incubated again at 60 C for 30 45 min to complete the A addition and or diluted The samples should be re typed NOTE Microvariants may differ by only one base pair so interpretation should be made with caution Microvariants will not change base pair value upon re incubation and retyping PULL UP Small artifactual peaks can appear in other colors under true peaks This phenomenon is termed pull up Pull up is the res
112. ITRATE pH 5 0 1L Dissolve 368 g trisodium citrate dihydrate Na3CgHs07e2H20 1600 ml ultrapure Adjust pH to 5 0 0 2 using approximately 60 g citric acid monohydrate 2 Bring up to 1 liter with ultrapure water mix autoclave Store at room temperature CITRATE BUFFER 0 1M SODIUM CITRATE pH 5 0 1L Add 100 10X Citrate Buffer to 900 ultrapure water Store at room temperature DEIONIZED FORMAMIDE 50 ml Mix 50 ml formamide and 5 g ion exchange resin Stir for 30 minutes at room temperature Check that the pH is greater than 7 If the pH is less than 7 decant the formamide into a beaker containing another 5 g ion exchange resin and repeat the 30 minute stirring at room temperature Allow the resin to settle Filter the formamide and aliquot into 1 5 ml vials Store up to 3 months at 20 C Optionally use manufactured pre deionized formamide aliquot and store at 20 C DIGEST BUFFER 10mM Tris HCl 10mM EDTA 50mM NaCl 2 SDS pH 7 5 100 ml Mix together 1ml 1M Tris HCl pH 7 5 2 0 5M EDTA 1ml 5M NaCl 10 ml 20 w v SDS and 86 ml sterile ultrapure water Store at room temperature Page 27 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 1M DITHIOTHREITOL 10mM SODIUM ACETATE pH 5 2 5 ml Dissolve 0 77 g dithiothreitol in 5 ml sterile ultrapure water Add 50 ul 1M sodium acetate pH 5 2 Do not
113. K SOLUTIONS Loc 1M TRIS HCL PH 7 5 MODEL Note Use an electrode suitable for measuring Tris buffers Adjust volume to 1 liter with ultrapure water Sterilize by autoclaving Store at room temperature Solution Preparation Dissolve 121 1 g Tris base in 800 ml ultrapure water Adjust pH to 7 5 0 2 using concentrated approximately 65 ml Date Prepared Amount Tris Base lot HCl concentrated lot Preparer pH TRISPH75 Page 229 of 255 Appendix V DNA Stock Solutions Log 1M Tris pH 7 5 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 APPENDIX W DNA STOCK SOLUTIONS Loc 1M TRIS HCL PH 8 0 MODEL Note Use an electrode suitable for measuring Tris buffers Adjust volume to 1 liter with ultrapure water Sterilize by autoclaving Store at RT Solution Preparation Dissolve 121 1 g Tris base in 800 ml ultrapure water Adjust pH to 8 0 0 2 using concentrated approximately 45 ml Date Prepared Amount Tris Base lot HCl concentrated lot Preparer pH TRISPH82 Page 230 of 255 DNA Stock Solutions Log 1M Tris pH 8 0 Model DNA Stock Solutions Log 1M Tris HCI pH 8 0 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY D
114. ML Solution Preparation UV treat all glassware and equipment Mix together 1ml 1M Tris HCl pH 7 5 2 0 5M EDTA 1 ml 5M NaCl 10 ml 20 w v SDS and 86 ml sterile ultrapure water Aliquot into 50 ml conical tubes Store at room temperature Bate Amount 1 Tris HCllot amp 0 5MEDTAlot 5MNaClloti 20 SDSlot Preparer venned Prepared initials date DIGBUFF Page 234 of 255 Appendix AA DNA Working Solutions Log Digest Buffer CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 APPENDIX AB DNA SOLUTIONS Loc 1M DTT DITHIOTHREITOL 10MM SODIUM ACETATE PH 5 2 MODEL 5 ML Solution Preparation Dissolve 0 77 dithiothreitol 5 ml sterile ultrapure water Add 50 uL 1M sodium acetate pH 5 2 Solution may be sterilized by filtration through a 0 2 0 45 micron filter Do not autoclave Aliquot recommended 0 5 ml and store at 20 C Date Prepared Amount Dithiothreitol lot 1 sodium acetate pH 5 2 lot Preparer Verified by initials date DTT Page 235 of 255 Appendix AB DNA Working Solutions Log 1M DTT Dithiothreitol 10mM Sodium Acetate pH 5 2 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 APPENDIX AC DNA WORKING SOLUTIONS Loc 200MM EDTA MODEL
115. N 2 1 JUNE 2002 SHORT TANDEM REPEAT ANALYSIS PRINCIPLES OF STR ANALYSIS CASEWORK AmpF STR Profiler Plus and AmpF STR Cofiler The AmpF STR Profiler Plus Profiler Plus or PP and AmpF STR Cofiler Cofiler or CO DNA typing systems Applied Biosystems utilize the polymerase chain reaction PCR to amplify regions of DNA known as short tandems repeats STRs in order to characterize DNA extracted from forensic specimens Profiler Plus and Cofiler are multiplex systems which allow for the simultaneous amplification of numerous STR loci as well as a portion of the amelogenin gene located within the X and Y chromosomes Analysis of amelogenin allows for gender determination The Profiler Plus and Cofiler kits contain all reagents needed for amplification which includes primer sets which are specific for the various loci PCR reaction buffer and AmpliTaq Gold DNA polymerase as well as the required allelic ladders The primer sets contained within each kit consist of both unlabeled primers and those that are labeled with one of three distinctive fluorescent dyes The use of multicolor dyes permits the analysis of loci with overlapping size ranges The amplified fragments are separated according to size by capillary electrophoresis using the ABI Prism 310 Genetic Analyzer The amplified fragments are detected by laser excitation A charged coupled device CCD camera displays the signals as peaks and then captures the subsequent emi
116. NA ANALYSIS SOP VERSION 2 1 JUNE 2002 APPENDIX X DNA WORKING SOLUTIONS Loc 5 W V CHELEX SOLUTION MODEL Solution Preparation Before starting UV treat all equipment and glassware When pipetting Chelex Stock Solutions the resin beads must be distributed evenly in solution this can be achieved by gently mixing with a stir bar in a beaker Also the pipette tip used must have a relatively large bore 1ml tips are adequate Add 5 g Chelex per every 100 ml sterile ultrapure water Aliquot into sterile 1 5 ml tubes Check pH of one of the tubes it should be greater than 9 Date Amount Chelex lot Preparer Verified by initials date repared CHELEX5 Page 231 of 255 Appendix X DNA Working Solutions Log 5 w v Chelex Solution CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 APPENDIX Y DNA WORKING SOLUTIONS Loc 20 w v CHELEX SOLUTION MODEL Solution Preparation Before starting UV treat all equipment and glassware When pipetting Chelex Stock Solutions the resin beads must be distributed evenly in solution this can be achieved by gently mixing with a stir bar in a beaker Also the pipette tip used must have a relatively large bore 1ml tips are adequate Add 20 g Chelex per every 100 ml sterile ultrapure water Aliquot into sterile 1 5 ml tubes Check pH of one of the tubes it s
117. NDIX P DNA SOLUTIONS Loc 5M NACL MODEL Solution Preparation Dissolve 292 g NaCl in 800 ml ultrapure water Adjust the final volume to 1 liter Sterilize by autoclaving Store at room temperature Date Prepared Amount NaCl Reagent Grade lot Preparer NACL2 Page 223 of 255 Appendix P DNA Stock Solutions Log 5M NaCl CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 APPENDIX Q DNA STOCK SOLUTIONS Loc PBS BurFER PH 7 4 PHOSPHATE BUFFER SALINE MODEL 2 7MM KCL 137MM NACL 1 5MM KH PO PH 7 4 1L Solution Preparation Dissolve 0 2 g KCI 8 0 g NaCl 0 2 g gt 1 1 of Na2HPO anhydrous 800 ml ultrapure water Adjust pH of solution to 7 4 if necessary Adjust to final volume to 1 liter using ultrapure water Sterilize by autoclaving Store at room temperature Date Prepared Amount KCI lot NaCl lot KH2PO lot Naz HPO lot Preparer Verified by initials date PBSBUFF Page 224 of 255 Appendix Q DNA Stock Solutions Log PBS Buffer pH 7 4 Phosphate Buffer Saline CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 APPENDIX R DNA STOCK SOLUTIONS Loc 1M SODIUM ACETATE 5 2 MODEL 100 ML Solution Preparation Dissolve 13 6 CH3COONa 3H 0 sodium acetat
118. NISTRATOR A The Local CODIS Administrator along with the Technical Leader is responsible for ensuring that the DNA section is in compliance with all aspects of the NDIS Standards for Acceptance of DNA Data including quality assurance of the data The duty requirements for the Local CODIS Administrator are as follows 1 The Local CODIS Administrator must possess a Bachelor s degree in a natural science or computer science 2 The Local CODIS Administrator shall have a working knowledge of computers computer networks and computer database management 3 The Local CODIS Administrator shall have an understanding of DNA profile interpretation Page 164 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 4 The Local CODIS Administrator functions as the system administrator of the laboratory s CODIS network and is responsible for all operations of the local CODIS system This responsibility includes but is not limited to a software updates security of the DNA profile data stored in CODIS training analysts in the use of the CODIS system tape backups of the system uploading DNA profiles into SDIS processing outside agency search requests CS Oa oe maintaining monthly hit records and forwarding them to the State CODIS Administrator h maintaining records as required by the State CODIS Administrator i ensuring that submitting agencies are notified
119. OR Mother 8 9 Child 8 9 Obligate alleles 8 and 9 If there is only one obligate allele use equation 2 However if there are two obligate alleles use equation 1 Again once the RPNE is determined for each locus then the PE for all loci would be determined If the questioned parent s do not share the obligate allele s with the child then an exclusion has occurred To determine that the parent s are excluded as possible biological parents there must be at least three 3 exclusions That is the child must not share an obligate allele with the parent s in at least three 3 different loci In these cases the parent s are excluded and no calculations are necessary Finally considering cases in which there are exclusions at only one 1 or two 2 loci The explanations for this occurring are as follows First is that the parent s is not the biological parent s and additional exclusions will be obtained when different systems are analyzed The second is that there has been a mutation s Finally a sibling of the suspected parent is the biological parent In these cases additional information may be necessary Refer to your AIC for current information as to how to handle cases with exclusions at only one 1 or two 2 loci Page 120 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 Kinship Relationship In some cases it may be necessary to evaluate by genetic testing that an individ
120. ORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 EQUIPMENT DNA THERMAL CYCLER 480 AND 9700 The DNA Thermal Cycler 480 or 9700 can be used to amplify both Chelex extracted and FTA extracted database samples The function of the thermal cycler is critical to the proper performance of the STR typing procedure thus the thermal cycler is calibrated semiannually See the User s Manual for each model instrument for further details on calibration ABI PRISM 377 DNA SEQUENCER The ABI Prism 377 DNA Sequencer is a fluorescent gel imaging system that is capable of determining base pair sequence fragment size or relative quantity of fluorescent dye labeled DNA nucleotide fragments It consists of an electrophoresis instrument with a laser excitation and CCD camera imaging system and a desktop computer with software for data collection and analysis Accessories such as gel cassettes glass plates and buffer reservoirs are included See the AB Prism 377 Sequencer User s Manual for further details ABI PRISM 3100 GENETIC ANALYZER The ABI PRISM 3100 genetic analyzer is an automated capillary array electrophoresis system This system can simultaneously separate detect and analyze fluorescent labeled DNA fragments from 16 capillaries in one run The system includes an ABI PRISM 3100 Genetic Analyzer and software computer workstation with Microsoft Windows NT operating system collection software capillary array and reagent consumab
121. ORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 or a pipettor with tips plugged with hydrophobic filters Pipet carefully and at a slight angle to minimize mixing and to avoid splashing the solution It is important to begin the cycling process within 20 minutes after the addition of the AmpliType Primer Set to the AmpliType PCR Reaction Mix to minimize the formation of primer dimer and other non specific PCR products G Carefully add 2 drops of mineral oil from the dropper bottle provided in the kit to all tubes including the controls before proceeding Be careful NOT to touch the reaction tubes with the dropper bottle Cap each tube loosely Do NOT vortex mix or spin NOTE Each AmpliType PM DQA1 PCR amplification is performed in a final volume of 100 ul Exactly 18 ul has been allocated for sample addition H For each of the following additions complete the processing of each tube before proceeding to the next tube No more than one tube should be opened at a time when making the following additions Use a new sterile pipette tip for each addition Open the tube and carefully add 18 ul of extracted Sample or Control DNA 1 Carefully insert the pipette tip through the mineral oil layer Discard the pipette tip and re cap the tube tightly before proceeding to the next sample Do NOT vortex mix or spin Prepare tubes as follows 1 DNA TEST SAMPLE TUBES add 18 ul of sample DNA to each labeled DNA test sample t
122. RENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 SALIVA STAINS OR SWABS Collect a portion of stain or swab for DNA extraction Place the material into a 2 2 ml microcentrifuge tube A To the sample add 300 ul organic stain extraction buffer 12 ul 1M DTT and 4 ul 10 mg ml Proteinase K solution Vortex for 1 second and spin in a microcentrifuge for 2 seconds to force the cutting into the extractions fluid B Incubate the tube at 56 C overnight C Spin in a microcentrifuge for 2 seconds to force condensation into the bottom of the tube D In a fume hood add 300 ul phenol chloroform isoamyl alcohol to the stain extract Vortex the mixture briefly to attain a milky emulsion Spin the tube in a microcentrifuge for 3 minutes Note If necessary additional organic extractions may be performed prior to the purification steps E Assemble a Microcon concentrator unit To the top of the concentrator add 100 ul Transfer the aqueous phase from the tube in Step D to the top of the concentrator Avoid pipetting organic solvent from the tube into the concentrator F Place a cap on the concentrator and spin in a microcentrifuge at 2500 x g for 10 minutes G Add 100 tl to the concentrator Replace the cap and spin the assembly in a microcentrifuge at 2500 x g for 10 minutes H Remove the cap and add a measured volume of between 20 ul and 200 yl to the concentrator Remove the concentrator from the
123. ROLS 115 INTERPRETATION OF THE SPECIMENS 116 SINGLE 116 EPI PRSE S 116 APPLICATION OF POPULATION FREQUENCY DATA TO PROFILER PLUS AND COFILER TYPING RESULTS S eese ess brass FERE EFI EE E Eo ers 16 117 CRIMINAL PATERNITY CASES Eee o petes 119 Kinship SLALOM ell 121 ELECTRONIC FILE NAMING CONVENTION FOR THE 310 STR PINAY SIS H non 121 REPORT WRITING S i UN ME MEIN EUM 122 CONTENTS OF PCR REPORTS 5 RES ES EDI e DIE D PIED PIED 122 GENERAL CATEGORIES OF TESTING CONCLUSIONS 122 POPSTA TS Z e inea dpud ix ea ad 123 Page 4 of 255 Table of Contents Continued CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 PRINCIPLES OF STR ANALYSIS 124 GUIDELINES FOR CONTROL 4444000 1 nennt nennen 128 EXTRACTION CONTROL Sint 4 5 ce Oo ca eec ieu dew oe de 128 AMPLIFICATION CONTROLS ics eeu otia s tini Foniee beer gai 128 EGUIPMISNIT s aeo et
124. RY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 6 Examine the Import Status results a Click on the printer icon b Choose screen E The action taken for a particular profile will be described red message indicates problems with the data T Close the Import Status results file 8 Exit from CODIS Import E The profiles are now in LDAS F Transferring Profiles from LDAS to LDIS 1 Log on to CODIS Local by entering the User ID and Password 2 Choose PCR Analysis by clicking on PCR 3 Click on Batch Transfer 4 Bring up imported profiles for transfer by choosing the following a Specimen Category Convicted Offenders b Assigned To Analyst s Name LDIS Filter None in LDIS for new specimens Some in LDIS for old specimens 5 Click Query A blue background should highlight all profiles 6 Check that all loci are marked with a white X G Click on Start Transfer All blue highlighted profiles will be transferred to LDIS H Close Batch Transfer l Exit from CODIS Local J The profiles are now in LDIS CODIS SEARCHES AND UPLOADS A If necessary a search of the database will be conducted before a DNA case is reported The DNA analyst who processed the case is responsible for initiating the search The results of this search should be reported to the local agency by way of the case report There are two mechanisms available for searching 1 Analysts may use Searcher under the State programs to search their Local G
125. RY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 Ladder Review the ladder to verify all peaks are called on the electropherogram Samples and Positive Extraction Control View samples individually and assess overall success of the injection Examine the following peak shape and height matrix quality peak profile size standard Make sure that the analysis parameter used captures 75 400 bp range and that the GeneScan size standard peaks are sized correctly Check that the Blue Green Yellow and Red peaks for each locus are all present In the Results Control window Verify GS size standard Precision With quick tile off select size standard color only can view up to 16 at a time Align by size and select the 250 basepair peak Show selected items only and verify that the 250 basepair calls are all within one basepair of each other Peak assignment Verify size standard peaks are assigned the correct types All peaks assigned a size should end in 00 i e 75 00 basepair Note It may be necessary to assign a new analysis parameter AP and or size standard for individual samples or ladders based on results obtained during analysis i e assignment of new AP to capture 75 400 bp range In addition database profiles are derived from single source samples Therefore the peak amplitude threshold may be lowered to 50 RFUs The technical leader must review and approve any allele calls below 50 RFUs J Af
126. Reagents are stored aliquots as small as practical This will allow for tracing of any contamination and minimize the number of times a given reagent is exposed to potential contamination Tubes containing DNA in liquid are centrifuged before opening Appropriate controls are always used At a minimum these controls include the positive amplification control the negative amplification blank and the appropriate reagent blanks and extraction controls casework e DNA from casework samples is ultimately stored in screw cap tubes Equipment centrifuges pipettors racks etc is cleaned as needed The PCR workstation is exposed to ultraviolet light for 20 30 minutes between preparation cutting removing etc of evidence samples and standard samples between extractions of samples with minimal amounts of DNA and samples with high levels of DNA between DNA extractions and PCR setup and before and after each use Glassware is cleaned thoroughly and or autoclaved prior to reagent storage Page 12 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 e The quantity of samples handled in a single run is limited to a manageable number This precaution reduces the risk of sample mix up and the potential for sample to sample contamination Clean dedicated lab coats are worn at all times PCR SETUP AREA PCR setup is performed in the PCR workstations located in th
127. Rinse thoroughly in ethanol and place onto clean paper Use a clean scalpel or clean scissors to cut a 1 cm portion from the root end of the hair Because hair may contain cellular material on the surface that may or may not originate from the hair donor it is advisable to cut off a 1 cm section of the shaft adjacent to the root portion for separate analysis as a control or 2 Place hair into a dish with sterile saline and while still immersed cut a 1 cm portion from the root end of the hair It is advisable to cut off a 1 cm section of the shaft adjacent to the root portion as a control O Add the root portion of the hair to 200 ul of 5 Chelex in a 1 5 ml microcentrifuge tube Add 2 ul 10 mg ml Proteinase Make sure that the root portion of the hair is the Chelex P Incubate at 56 C at least 6 to 8 hours overnight o Vortex at high speed for 5 to 10 seconds R Spin for 10 to 20 seconds in microcentrifuge at 10 000 to 15 000 x g NOTE Check that the hair root is completely immersed in the Chelex solution before boiling Incubate in a boiling water bath for 8 minutes Vortex at high speed for 5 to 10 seconds Spin in a microcentrifuge for 2 to 3 minutes at 10 000 to 15 000 x g The sample is now ready for the PCR Amplification Process Store the remainder of the supernatant at either 2 to 8 C frozen To re use repeat Steps I through K CONCENTRATION OPTION Concentration by Micro
128. SIS SOP VERSION 2 1 JUNE 2002 ABI 373 and ABI PRISM 377 DNA Sequencers GeneScan Reference Guide 1997 ABI PRISM 3100 Genetic Analyzer User s Manual 2001 ABI PRISM GenoScan Analysis Software 2 1 User s Manual Sept 1996 ABI PRISM GeneScan Analysis Software Version 3 7 for the Windows NT Platform User s Guide 2001 ABI PRISM 2 0 User s Manual 1996 ABI PRISM GenoTyper 3 7 NT Software User s Manual 2001 AmpF STRQO Cofiler PCR Amplification Kit User Bulletin 1998 AmpF STRO Identifiler Users Manual 2001 AmpF4STR Profiler Plus PCR Amplification Kit User s Manual 1998 AmpF STRG Profiler Plus User s Manual 1998 and AmpFISTR CofilerTM User s Manual 1998 Anker R Steinbrueck T and Donis Keller H Tetranucleotide Repeat Polymorphism at the Human Thyroid Peroxidase hTPO Locus Hum Mol Gen 1 2 1992 137 Automated Geno Typing Protocol Armed Forces DNA Identification Laboratory Aug 1998 Budowle B Moretti T Keys K Koons B and Smerick J Validation Studies of the CTT Multiplex System J For Sci 42 1997 701 7 Buel E Schwartz and LoFountain M Capillary Electrophoresis STR Analysis Comparison to Gel Based Systems J For Sci 43 1998 164 70 Clark J Novel Non templated Nucleotide Addition Reactions Catalyzed by Procaryotic and Eukaryotic DNA Polymerases Nucleic Acid Res 16 1988 9677 86 Crouse C A and Schumm J I
129. SUES Tissues should be stored at 20 C until used A Place a portion of the tissue on a clean surface Remove a piece of tissue approximately 1 B Mince tissue into small pieces and place in a 2 2 ml or 1 5 ml microcentrifuge tube To the sample add 300 yl organic stain extraction buffer 12 ul 1M DTT and 4 ul 10 mg ml Proteinase K solution Vortex for 1 second and spin in a microcentrifuge for 2 seconds to force the sample into the extraction fluid D Incubate the tube at 56 C overnight E Spin in a microcentrifuge for 2 seconds to force condensation into the bottom of the tube In a fume hood add 300 ul phenol chloroform isoamyl alcohol to the extract Vortex low speed the mixture briefly to attain a milky emulsion Spin the tube in a microcentrifuge for 3 minutes Note If necessary additional organic extractions may be performed prior to the purification steps G To a Microcon concentrator add 100 ul Transfer the aqueous phase from the tube in Step F to the concentrator Avoid pipetting organic solvent from the tube into the concentrator H Place a cap on the concentrator and spin in a microcentrifuge at 2500 x g for 10 minutes l Add 100 ul to the concentrator Replace the spin cap and spin the assembly a microcentrifuge at 2500 x g for 10 minutes J Remove the and add a measured volume of TE that is between 20 ul and 200 ul to the concentrator Remove the concent
130. Sheet and Plate Record Excel format save the 3100 load sheet as a tab delimited text file and save the Excel worksheet separately 9 Import the plate record Import the plate record into the 3100 collection software and check to make sure that it has been imported correctly Do this by highlighting the plate record and clicking the edit button Make sure the columns are all filled in and that the injection number is correct Refer to Page 142 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 Chapter 6 of the ABI 3100 User s Manual for more information regarding plate records Sample preparation Through instrument and PCR kit validation studies each laboratory has evaluated their own methods and DNA quantities used in sample preparation This includes the amount of formamide ROX or LIZ and PCR product placed into each well on the 3100 optical plates Each well should contain a minimum of 10ul of the combined components After all samples are loaded onto the well plates the samples are covered with a 96 well plate septa gasket 1 Remove deionized formamide from the freezer to thaw 2 Add the internal size standard to the deionized formamide and pipet this mixture into the wells of the 96 well plate 3 Pipet the PCR products into the well determined by the plate record making sure to include one ladder per set of 16 wells 4 Secure the septa to the plate 5 Denature
131. TABASE b Ee come cost 177 SUBMISSION PREPARATION AND STORAGE OF SAMPLES FOR OFFENDER DATABASE ri enc re crt m trud crt poten 177 CBI HANDLING OF DATABASE SAMPLE SUBMISSIONS 178 APPENDIX tree 181 ANALYST ACTIVITY AA FORMS iecit e ape Rea pen 183 TERMS AND DEFINITIONS FOR CODIS nene 185 RESOURGBS3o a a dta utu ste e ID C E 189 GENERAL RESOURCES Dati nd 189 AMELOGENIN S hec btt dad 189 Geo Secun Moto OI DN DM ND RN 190 DNA COUANTIEICATIONS ei ERI CHEER IDEE ED DR EE FEDERE 190 POLYMERASE CHAIN REACTION 190 STRANALYSIS a cre edicit de ceci edd t c e edt dies 191 AM LI MINIME IN IDE 195 ALLELE FREQUENCIES OR 197 T3595 boue die e o etie MEC MEL ED EE 197 c E x CS 198 Page 7 of 255 Table of Contents Continued CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 PGA coe cen ee 199 DSS 1170 200 OTSA tek fae testa M
132. TABASE DNA ANALYST 4 cnt oc e een 164 LOCAL LDIS CODIS ADMINISTRATOR 220000018 164 Page 6 of 255 Table of Contents Continued CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 STATE SDIS CODIS 165 DNA PROFILE 166 APPROVED DNA PROFILES FOR 5 4 166 SAMPLE INPUT PROTOCOL 11 167 SAMPLE INPUT PROTOCOL DATABASE 169 CODIS SEARCHES AND UPLOADS 170 COMMUNICATION PROTOCOL FOR A POSSIBLE CODIS HIT 171 DELETING CODIS PROFILES 173 REQUESTS FOR EXPUNGEMENT 4 eU E E A TERES I EA 173 NDIS QUALITY ASSURANCE QUALITY CONTROL STANDARDS 174 PROFICIENCY TESTING zucca DU ALII etn ve du us 174 AUDITS An tend sten n EET cn roh cre 175 SYSTEMS OPERATIONS NUM x 175 GODIS SECURITY ic E EU cae eee 175 TAPE BACKUP PROCEDURES eut v RECO x uUo is Ee cma 175 PILE STORAGE Sab DU EE DE 175 DNAJDA
133. Transfer 120 ul DNA Standard A into the tube labeled d Aliquot 60 ul TE Buffer into each of the six remaining tubes labeled through G e Add 60 yl DNA Standard A tube A to the 60 ul TE Buffer in tube B Vortex to mix thoroughly f Add 60 ul diluted DNA Standard B tube B to the 60 ul TE Buffer in tube C Vortex to mix thoroughly g Add 60 ul diluted DNA Standard C tube C to the 60 ul TE Buffer in tube D Vortex to mix thoroughly h Continue the serial dilution through tube G i If the dilution steps are performed as described in Section 2 c h above the seven DNA Standard tubes tubes A through G will have the concentrations of human DNA listed in Table 1 Page 56 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 Table 1 DNA Standards and Concentrations ng ul ng NOTE Store the diluted DNA Standards at 2 to 8 C The DNA Standards through G are stable for at least three months at 2 to 8 C B Information Regarding Protocols e N This entire section should be read before slot blotting immobilization of DNA The QuantiBlot Human DNA Quantitation Kit contains reagents for at least 10 hybridization reactions Each hybridization reaction should include the following ten control samples seven DNA Standards the two DNA Calibrators and one blank Spotting Solution only An additional 38 sampl
134. Type HLA DQA1 DNA Probe Strip 13 All but 1 3 signal weaker than C dot on AmpliType HLA DQA1 DNA Probe Strip REFERENCES JUNE 2002 POSSIBLE CAUSE EDTA not added to the reaction prior to the heat denaturation step of the DNA hybridization pro tocol EDTA not added to the reaction prior to the heat denaturation step of the DNA hybridization proto col Genotype of sample has a HLA DQA1 4 2 or 4 3 allele paired with a HLA DQA1 1 1 2 3 4 2 or 4 3 allele Amplification of DQA2 pseudogene faint 1 1 dot Genotype of sample has a HLA DQA1 1 3 allele paired with a HLA DQA1 4 1 4 2 or 4 3 allele RECOMMENDED ACTION Add EDTA to amplified sample repeat test Add EDTA to amplified sample repeat test See Reference 18 See References 18 and 30 See Reference 18 T von Beroldingen C H Blake E T Higuchi R Sensabaugh G F Erlich H A Applications of PCR to the Analysis of Biological Evidence PCR Technology Principles and Applications for DNA Amplification Ed Erlich H A New York NY Stockton Press Inc 1989 209 23 2 Reynolds R Sensabaugh G and Blake E Analysis of Genetic Markers in Forensic DNA Samples Using the Polymerase Chain Reaction Analytical Chemistry 63 1991 2 15 Page 88 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 Saiki R K Gelfand D H Stoffel S Scharf S J Hi
135. USING ERIE ABI S se eek e 134 POURING A POLYACRYLAMIDE GELD o e e en e e e ec 134 MACINTOSH PLATFORM PROCEDURE 135 STR TYPING BY CAPILLARY GEL ELECTROPHORESIS USING THE ABI PRISM 3100 GENETIC 139 GENESCARN ANALYSIS re eoe Dr eret Di o Eo Ht Ea C abere etos 145 GENESCAN ANALYSIS USING MACINTOSH 145 GENESCAN ANALYSIS USING NT SOFTWARE 147 GENOTYPER JANALENY ice eda 149 Page 5 of 255 Table of Contents Continued CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 USING A TEMPLATE FILE CO RI D AR Gr e E EYE EX EROS 150 EXAMINING DATA dad diede ar coe tota ec 151 INTERPRETATION OF DATA edite us ntes ee 152 5 5 T TEST 152 EVALUATION OF STR DATA ct iS E AN DEN E CU DN LER EN QUEEN BARRERA 152 dadocd odes eb dob te toda oett e dc PO os 153 NON TEMPLATE NUCLEOTIDE ADDITION 153 Vl ota Po go CR MCN 154 SPIKES AND OTHER ANOMALIES 2 va aL ES MEE onan 154 enit etus 154
136. Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 B Select OK to execute merging of specimens Specimen details are as follows Specimen Category Convicted Offender Tissue Type Blood Tissue Form Stain Population group Unknown Click box Use these settings for every specimen C Save the report that is generated as REPORTdateana Print the report for the review and sample records Document the rejected samples Initial any concerns D Transfer the CMF file to a CODIS computer by way of the shared folder on the CODIS server CREATING A CMF FILE USING MACINTOSH SOFTWARE The DataBankSTR software functions solely to convert Excel files into a common message format which can then be transferred into CODIS using the CODIS Import software Therefore the technical review of the profiles is accomplished as a separate process A Create a CODIS Genotyper file for the PP and CO runs 1 Open the appropriate Genotyper file GT mm dd yy anaPP and save it as a CODIS GT file CODIS GT mm dd yy anaPP 2 Edit the CODIS GT file by removing all ladders and control samples Leave only the DNA profiles to be imported into CODIS 3 Run the Make Codis Table macro command 6 Export the resulting table as CODIS GT mm dd yy anaPP to the appropriate Run Folder 4 Once all CODIS GT tables are created and exported exit the Genotyper software B Import profiles into DataBankSTR 1 Launch DataBankSTR and
137. Wash Solution solids must be completely dissolved and well mixed before use O Dispense 5 ml pre warmed PM DQA1 Wash Solution into each well a dispensing re pipette is useful for this purpose Rinse by rocking the tray for several seconds then aspirate the solution from each well Remove any solution from the tray lid with a clean lab wipe P Dispense 3 ml of the Enzyme Conjugate Solution prepared in step L into each well and cover with the clear plastic lid Transfer the tray to the 55 C water bath Place a 1 kg weight on the covered tray to prevent the tray from sliding or floating Adjust the rotating water bath to 50 to 90 rpm Check the tray position and insure that water does not splash into the wells of the tray Page 75 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 Q Replace the water bath cover to maintain the temperature at 55 C 1 C Incubate the Enzyme Conjugate Solution with the DNA probe strips at 55 C 1 C for 5 minutes R After incubation remove the tray from the water bath tip tray at a slight angle and aspirate the contents of each well from the labeled end of the strips Remove condensation from the tray lid with a clean lab wipe NOTE Replace the water bath cover to maintain the temperature at 55 C 1 Keep the water bath rotating between incubation steps 5 Dispense 5 ml pre warmed PM DQA1 Wash Solution into each well Rinse by
138. With Known Contributor In instances where a contributor is known or expected to be present e g certain sexual assault samples assignment of the known contributors alleles may allow for the determination of the remaining profile and a frequency may be reported Indistinguishable Mixtures If a major and minor contributor cannot be distinguished because of similarity in peak heights or the presence of overlapping alleles that cannot be assigned individuals may or may not be included or excluded and a combined genotype frequency may be determined using the mixture calculation defined below In this instance all alleles greater than or equal to 50 RFU will be included in the calculations APPLICATION OF POPULATION FREQUENCY DATA TO PROFILER PLUS AND COFILER TYPING RESULTS The Allele Frequencies Appendix lists the Profiler Plus and Cofiler STR allele frequencies for three population African American Caucasians and SW Hispanics Additionally other databases are available in the literature if needed Genotype frequencies obtained for DNA alleles at one locus can be multiplied by the frequencies found for the same sample at the other twelve loci to obtain a combined frequency estimate Heterozygote frequencies will be calculated using the formula f 2pq Homozygous frequencies will be calculated using the NRC II formula P 1 0 with 0 01 Page 117 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA AN
139. air into the POP 4 will dissolve the urea If the precipitate will not dissolve do not use the POP 4 Check that the POP 4 is specifically for use with the 3100 Set up the instrument 1 Prime the syringes Draw up filter purified water into the syringes and then expel Draw up approximately 0 2 ml of room temperature POP 4 into the reserve syringe Pull plunger so that the tip is drawn to the position where the polymer will be filled Remove air bubbles from the tip and slowly expel the POP 4 onto a Kimwipe or waste container Follow the same procedure with the capillary array syringe using approximately 0 1 ml of POP 4 2 Fill both the reserve syringe and the capillary array syringe with room temperature POP 4 Try to minimize the amount of air bubbles in the syringes The polymer is good at 25 C for 7 days and should be changed weekly Page 139 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 3 The approximate amount of polymer needed for a run can be calculated by taking the of injections x 50 80 ul POP 4 200 ul POP 4 which is required to fill the blocks and tubing 4 Clean the outside of the syringe with distilled water then dry with a Kimwipe to remove excess polymer 5 Remove any air bubbles by inverting the syringe and pushing out a small amount of polymer NOTE It is critical that large air bubbles be expelled from the syringe M
140. ake sure the plunger is not resting on a bed of air as this may cause problems in the run 6 Before installing the capillary array launch the Install Capillary Array wizard from the Tools menu on the 3100 collection software Follow the directions given in the wizard to replace or install an array Install the array and the syringes filled with polymer After a few times going through the wizard it may not be necessary to launch the wizard every time you reinstall an array However if you are replacing an array with a new one make sure you run the wizard to log the serial number of the new array Note It is not necessary to change the capillary array for every run A capillary array should last a minimum of 100 runs The maximum of injections per capillary is approximately 300 T Fill buffer and water reservoirs Note that the 1X buffer and DI water should be changed daily or before each run The 1X buffer is made from the 10X buffer purchased from ABI for use on the 3100 and 310 instruments for fragment analysis NOTE A larger volume of 1X buffer can be made if desired The 1X solution can be stored at 4 C for up to 1 month 8 Place the 1X buffer reservoir in the 1 position cathode buffer on the tray rack and water reservoirs in the 2 and 4 positions Fill the anode buffer reservoir with 1X buffer to the red line and place it onto the lower polymer block 9 Perform a spatial calibration This should be per
141. al of Forensic Sciences 39 1994 41 51 Comey C T Koons B W Presley K W Smerick J B Sobieralski C A Stanley D M and Baechtel F S DNA Extraction Strategies for Amplified Fragment Length Polymorphism Analysis Journal of Forensic Sciences 39 1994 1254 9 Cosso S Reynolds R unpublished data RMS Page 91 of 255 CBI DNA Analysis SOP Vers 2 1 ALLELE FREQUENCIES DQA1 PM CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 Table 2 HLA DQA1 observed allele frequencies in several United States general population groups African Americans Caucasians Southwestern Hispanics 491 N 363 N 333 Allele 1 1 0 127 0 153 0 122 Allele 1 2 0 292 0 198 0 131 Allele 1 3 0 046 0 065 0 042 Allele 2 0 102 0 152 0 111 Allele 3 0 109 0 174 0 230 Allele 4 1 0 202 0 234 0 255 Allele 4 2 4 3 0 122 0 025 0 110 N refers to the number of individuals in the database Page 92 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 Table 3 Observed allele frequency distributions for PM loci in several United States general population groups African American Caucasian Southwestern Hispanic Allele LDLRA 0 192 0 432 0 535 N refers to the number of individuals in the database Page 93 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSIO
142. alDTABLE GTdateanalDana2TABLE REPORTdateana dateanaCMF Each database analyst will back up his her data for each month on two duplicate CD ROMs The following should be included for each set of samples to include all information from both the analyst and the technical reviewer s review of the data For the ABI 377 instrumentation Run folders Genotyper files CODIS Genotyper files Genotyper tables CODIS Genotyper tables DataBankSTR files Page 160 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 DataBankSTR CMF files Sample sheets Run notes GeneScan matrix files GeneScan standard files GeneScan parameter file For the ABI 3100 instrumentation Run folders GeneScan projects Genotyper files contains allele table CODIS Genotyper files CODIS Genotyper tables conversion reports CODIS excel cmf files Plate records Run notes Spectral and spatial calibrations GeneScan standard files GeneScan parameter files REFERENCES Page 161 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 This Page Intentionally Left Blank Page 162 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 CODIS INTRODUCTION TO CODIS CODIS stands for COmbined DNA Index System The purpose of CODIS is to create a national information repository wher
143. alleles or none of the alleles in the mixture profile will be entered into LDIS Elimination standards or forensic unknowns matching elimination standards will not be entered into CODIS Victim standards or forensic unknowns matching victims will not be entered into CODIS Suspect standards will not be entered into CODIS DNA analysts will use their discretion in regard to any other DNA profiles that might be generated during the course of their analysis that do not fit into any of the above categories e g a DNA profile from a bloodstain that may or may not have anything to do with the crime Off ladder alleles that vary by less than the consensus repeat unit will be designated as an integer of that variation 1 2 3 or X may be used for entry into CODIS Example 01 8 2 allele If an allele falls above the largest or below the smallest peak of the ladder of the NDIS acceptable alleles the allele will be designated as either greater than gt or less than lt the respective ladder allele for entry into CODIS For NDIS acceptance profiles derived from forensic unknown samples must have a minimum of 10 of the CODIS core STR loci Profiles from convicted offender samples must have all 13 CODIS core loci SAMPLE INPUT PROTOCOL CASEWORK A A forensic case may have more than one sample with the same DNA profile A single profile will be entered for each different DNA profile obtained in the case The first exhibit
144. aluated biannually ABI PRISM 310 Genetic Analyzer The ABI Prism 310 Genetic Analyzer is an automated capillary electrophoresis system This system can separate detect and analyze fluorescent labeled DNA fragments The system includes an ABI prism Genetic Analyzer collection software and computer workstation with MAC operation system and GeneScan analysis software Matrix A new matrix will be created for each instrument whenever analytic data indicates the necessity on a semiannual basis at minimum Refer to How to Create the GeneScan Matrix File in the GeneScan Analysis Software Experiments of the PRISM 310 Genetic Analyzer User s Manual for instructions on how to build a matrix Once prepared a matrix will be considered suitable for casework if a flat baseline is obtained when it is applied to the matrix standards NOTE A new matrix must be built following cleaning maintenance or replacement of a component of the optics Page 97 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 Capillary A new capillary will be installed whenever analytic data indicates the necessity approximately 300 injections Buffer and Waste Vials The cathode buffer and waste vials will be replaced at a minimum every 72 hours Polymer The POP 4 will be replaced at a minimum every 72 hours Power Macintosh Computers Hard drives will be defragmented periodically us
145. amelogenin D8 D8S1179 D19 D19S433 D2 D2S1338 P U pullup st stutter 7 decrease spk spike SO spill over RB reagent blank NAB negative amplification blank PC positive control Page 254 of 255 Appendix AU Genescan GenoTyper Review Sheet Model CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 APPENDIX AV 3200 LOAD SHEET MODEL Plate Name 3 4 A B D E F G H A B C D E F G H Page 255 of 255 Appendix AV 3200 Load Sheet Model CBI DNA Analysis SOP Vers 2 1
146. antification and Amplification process Ww UT Store the remainder of the supernatant at 2 to 8 C or frozen To re use repeat Steps through Page 33 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 SPERMATOZOA EXTRACTION IN THE PRESENCE OF CONTAMINATING CELLS POST COITAL A Dissect swab or fabric Use a clean cutting surface for each different sample Steps B through G optional B Pipet 150 to 500 ul of PBS into a sterile 1 5 ml microcentrifuge tube Add swab or fabric cutting C Incubate at 4 C for 2 hours or overnight D Twirl the swab or fabric with a sterile pick applicator stick for at least 2 minutes to agitate the cells off the substrate Remove the swab or fabric with the pick It is advisable not to discard the substrate until microscopic analysis Step P shows that the sample contains sperm Store swab or fabric in a sterile tube OPTIONAL Transfer swab or fabric into a Costar spin basket insert Place the basket insert into the tube containing the stain extract Spin in a microcentrifuge at maximum speed for five minutes Remove basket insert from extract tube Remove the extracted material from the basket place in a clean microfuge tube and freeze if required Otherwise discard F Centrifuge the sample in a microcentrifuge for 1 minute at 10 000 to 15 000 x g at room temperature A Costar spin basket may be utilized for the agitation
147. apillary into two tubes of buffer or filter purified water store until used again and d turning off the computer 7 If running additional samples replace the buffer and water vials if necessary gt 72 hours b replace the water waste tube and add POP 4 if necessary GENESCAN GENOTYPER ANALYSIS GeneScan analysis software is used to analyze the raw data collected by the CE310 Genetic Analyzer Genotyper software is used to automatically convert allele sizes from GeneScan Analysis software into allele designations and to build tables containing Genotyper information as needed Genotypes are assigned by comparing the sizes obtained for the unknown sample alleles with sizes obtained for the alleles in the allelic ladder Page 109 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 Utilizing the GeneScan and Genotyper analysis software determine the allele designation for each sample The following criteria must be accomplished during the use of the GeneScan and Genotyper analysis software refer to the appropriate software manuals as needed A B C K L Apply correct matrix Insure that red dye color is marked as the size standard Define and apply a size standard file with the peaks for 75 100 139 150 160 200 300 340 350 and 400 Profiler Plus base pair peaks labeled Define and apply an analysis parameter with the following criteria 1
148. autoclave Aliquot recommended 1 ml and store at 209 0 5M EDTA pH 8 0 1L Slowly add 186 1 g disodium ethylenediaminetetraacetic acid dihydrate Na EDTA 2 to 800 ml ultrapure water Stir vigorously on a magnetic stirrer Adjust to pH 8 0 by adding NaOH pellets approximately 20 g Adjust final volume to 1 liter with ultrapure water Sterilize by autoclaving Store at room temperature NOTE EDTA will not go into solution without pH adjustment 200mM EDTA 10 ml Add 4 0 ml 0 5M EDTA pH 8 0 to 6 0 ml of sterile ultrapure and mix thoroughly The solution may be aliquotted 300 jl tube into microcentrifuge tubes Store at 2 to 8 C HYBRIDIZATION SOLUTION 5X SSPE 0 596 w v SDS 1 L Add 250 ml 20X SSPE and 25 ml 20 w v SDS to 725 ml ultrapure water and mix thoroughly Hybridization Solution solids must be in solution before use warm in an incubator to 50 55 C to dissolve solids completely prior to use Preparation in a clear glass container is recommended to facilitate visual inspection for solids during warming 3 HYDROGEN PEROXIDE 1 ml Add 1 ml 30 to 9 ml sterile ultrapure water and vortex to mix Protect from light Store at 2 to 89 The 3 hydrogen peroxide has a shelf life of approximately 4 weeks 30 H2O 5 ml Aliquots Aliquot 5 ml into 15 ml conical tubes wrap with Parafilm and store at 4 C in QuantiBlot area ORGANIC SPERM WASH BUFFER 10mM Tris HCI 10mM EDTA 50
149. boratories established by local law enforcement agencies may participate in CODIS as an LDIS location Each laboratory must adhere to the NDIS Standards for Acceptance of DNA Data to become eligible They must apply through the State Administrator to the FBI If approved the FBI will install CODIS and that laboratory may upload forensic profiles to SDIS ORGANIZATION AND MANAGEMENT FORENSIC DNA ANALYST A D The forensic DNA analyst generates DNA profiles from forensic cases and determines whether or not a DNA profile obtained from a particular case will be entered into LDIS The analyst communicates his her determination through a copy of the final report and an appropriately marked and technically reviewed STR summary sheet Each forensic analyst must enter the appropriate profiles from his her case into CODIS The audit trail created during data entry must indicate that the person entering the profile s is the person who performed the analysis It is the responsibility of the analyst to notify the CODIS Administrator of any CODIS related actions on their casework DATABASE DNA ANALYST A B The database DNA analyst generates DNA profiles from the convicted offender cases and upon the completion of a technical review enters those profiles into LDIS It is the responsibility of the analyst to notify the CODIS Administrator of any CODIS related actions on their Convicted Offender Database samples Loca 1015 CODIS ADMI
150. by the analysts to aid in their Page 110 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 interpretation of the data presented them These criteria are based upon validation studies literature and casework experience These guidelines are not a set of hard and fast rules but rather a means to establish a general framework and outline minimum standards to insure that A conclusions in the casework report are scientifically supported by the analytical data including that obtained from appropriate standards and controls B interpretations are made as objectively as possible and C interpretations are consistent from analyst to analyst EVALUATION OF STR DATA The goal of the evaluation and interpretation of the STR data is to determine the DNA profiles developed from the analysis of case samples and comparison of these profiles with those from known individuals The following should be applied to aid in that interpretation A peak is defined as a triangular section of the electropherogram that results from the sum of input signals and as such shows Gaussian distribution The internal lane standard GS 500 ROX must have the correct sizes assigned to the peaks used for sizing It is noted that the 250 bp is not used for sizing purposes The 75 and 350 bp peaks must be captured for all samples The 400 bp peak must be captured for the 018551 allele or other alleles greater than
151. can be amplified Store the samples at 4 C or frozen Prior to the use of samples after storage they should be vortexed briefly and spun in a microcentrifuge for 5 seconds Page 40 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 ORGANIC EXTRACTION All extraction steps must be performed in the DNA extraction PCR setup laboratory using hoods reagents and pipettors dedicated to this area WHOLE BLOOD OR BLOODSTAINS Liquid blood samples should be made into bloodstains Stains should be air dried before being stored in individual plastic bags at 20 C until used A Place the bloodstain approximately 3 mm by 3 mm or 3 ul whole blood in a 2 2 ml microcentrifuge tube B To the sample add 300 ul organic stain extraction buffer 12 ul 1M DTT and 4 ul of 10 mg ml Proteinase K solution Vortex on low speed for 1 second and spin in a microcentrifuge for 2 seconds to force the cutting into the extraction fluid C Incubate the tube at 56 C overnight D Spin in a microcentrifuge for 2 seconds to force condensation into the bottom of the tube In a fume hood add 300 ul phenol chloroform isoamyl alcohol to the stain extract Vortex low speed the mixture briefly to attain a milky emulsion Spin the tube in a microcentrifuge for 3 minutes Note If necessary additional organic extractions may be performed prior to the purification steps Microcon conce
152. centrator unit To the top of the concentrator add 100 ul Transfer the aqueous phase from the tube in Step to the top of the concentrator Avoid pipetting organic solvent from the tube into the concentrator H Place a cap on the concentrator and spin a microcentrifuge at 2500 x g for 10 minutes l Add 100 to the concentrator Replace the cap and spin the assembly in a microcentrifuge at 2500 x g for 10 minutes J Remove the and add a measured volume of that is between 20 ul and 200 ul to the concentrator Remove the concentrator from the filtrate cup and carefully invert the concentrator onto a labeled retentate cup Discard the filtrate cup Note Final recovery volumes following purification generally range from 20 ul to 200 ul for evidentiary samples The final recovery volumes for the extraction reagent blanks for evidentiary samples are generally between 20 ul and 200 ul K Spin the assembly in a microcentrifuge at 2500 x g for 5 minutes L Discard the concentrator Cap the retentate cup Page 46 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 M Estimate the quantity of DNA in the sample by slot blot hybridization N After quantification the sample can be amplified O Store samples at 4 C or frozen Prior to the use of samples after storage they should be vortexed briefly and spun in a microcentrifuge for 5 seconds TIS
153. cler at 10 C until removed They can then be stored at 2 6 C for a week or longer at 15 to 20 C until processed and analyzed on the 310 SAMPLE PREPARATION A Determine the number of samples to be amplified Place the required number of amplification reaction tubes into a rack and label them B Fill out a STR dilution sheet C Samples will be diluted as necessary according to instrument sensitivity Twenty microliters of this solution will be added to the Profiler Plus Master mix in the appropriate amplification tube and another 20 yl will be added to the Cofiler Master Mix Page 99 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 AMPLIFICATION PREPARATION The following formulation applies to both the Profiler Plus and Cofiler kits A Vortex at medium speed PCR reaction mix primer set AmpliTaq Gold DNA polymerase reagents supplied in the kit for 5 seconds Spin the tubes briefly in a microcentrifuge to remove any liquid from the caps In 1 5 ml microcentrifuge tube put 1 Number of samples 1 x 21 0 ul of PCR reaction mix 2 Number of samples 1 x 1 0 ul of AmpliTaq Gold DNA polymerase and 3 Number of samples 1 x 11 0 ul of primer set The above formulation provides a slight overfill to allow for volume lost in pipetting The maximum volume of Master Mix held in a 1 5 ml tube can be dispensed into 42 PCR tubes A 2 0
154. collected in accordance with current Colorado statutes The following standards shall be used to determine if a profile qualifies for LDIS SDIS database entry A Forensic unknown samples recovered from evidence in a case where there is no identified suspect non suspect cases B Forensic unknown samples which have been compared to a suspect identified in the case regardless of whether the suspect is included or excluded C Profiles obtained from convicted offenders meeting current statutory requirements for inclusion in the Convicted Offender Database D Forensic unknown mixtures Page 166 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 1 If a mixture occurs that can be easily separated as known and unknown contributors only the alleles consistent with the unknown individual s will be entered into LDIS For example a vaginal swab that has a 4 allele pattern in which 2 of the alleles are consistent with the victim in the case The alleles consistent with the victim may be removed from the mixture leaving the remaining alleles to be entered into LDIS 2 Once the alleles from the known contributor are removed from the profile resulting single source DNA profiles may be entered into the Forensic Unknown Database 3 For more complicated mixtures care must be taken to determine which alleles if any should be entered into CODIS There may be instances in which all
155. con ultrafiltration may be recommended for Chelex extracted samples containing less than 500 ng of DNA Before discarding supernatant from the centrifugations it may be advisable to keep all until the analysis is complete The aqueous portion of a DNA extract may be concentrated as follows Page 39 of 255 CBI DNA Analysis SOP Vers 2 1 TG um CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 Assemble a Microcon ultrafiltration concentrator unit To the top of the concentrator add 100 ul TE Buffer Transfer the aqueous phase from the DNA extract to the concentrator Place cap on the concentrator and spin in a microcentrifuge at 2500 x g for 10 minutes OPTIONAL Carefully remove the concentrator unit from the assembly and discard the fluid from the filtrate cup Return the concentrator to the top of the filtrate cup Add 200 TE Buffer to the concentrator Replace cap and spin microcentrifuge at 2500 x g for 10 minutes Remove the cap and add a measured volume of TE Buffer that is between 20 yl and 200 yl to the concentrator Remove the concentrator from the filtrate cup and carefully invert the concentrator into a labeled retentate cup Discard the filtrate cup Spin the assembly in a microcentrifuge at 2500 x g for 5 minutes Discard the concentrator Cap the retentate cup If not previously done estimate the quantity of DNA by slot blot hybridization After quantification the sample
156. crocentrifuge to remove any liquid from the caps In a sterile microcentrifuge tube add 1 Number of samples x 21 0 ul of PCR reaction mix 2 Number of samples x 1 0 ul of AmpliTaq Gold DNA polymerase 3 Number of samples x 11 0 ul of primer set The above formulation provides a slight overfill to allow for volume lost in pipetting The maximum volume of Master Mix held in a 1 5 ml tube be dispensed into 42 PCR tubes A 2 0 ml tube is recommended when preparing Master Mix for up to 55 samples Be sure to include enough Master Mix for a reagent blank a positive amplification control and a negative amplification blank Mix thoroughly by vortexing at medium speed for 5 seconds Spin the tube briefly in a microcentrifuge to remove any liquid from the cap Sample preparation is detailed below Final amplification volume is 50 ul The following formulation applies to the Identifiler kit G Vortex the PCR reaction mix primer set and AmpliTaq Gold DNA polymerase supplied in the kit for 5 seconds at medium speed Spin the tubes briefly in a microcentrifuge to remove any liquid from the caps Page 131 of 255 CBI DNA Analysis SOP Vers 2 1 J K Ls CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 In a sterile microcentrifuge tube combine 1 Number of samples 1 x 10 5 uL of PCR reaction mix 2 Number of samples 1 x 0 5 uL of AmpliTaq Gold DNA polymerase and 3 Number of samples 1 x
157. cts GUIDELINES FOR CONTROL SAMPLES It is imperative that proper control samples be included when evidence samples are extracted quantified amplified and typed through capillary electrophoresis The typing results obtained from these control samples are essential for the interpretation of STR and amelogenin typing results from evidentiary samples The controls also serve to assess the quantity and quality of the extracted DNA as well as the effectiveness accuracy and precision of the analytical procedures EXTRACTION CONTROLS Reagent Blank With each set of extractions of evidentiary samples a reagent blank must be included The reagent blank consists of all reagents used in the test process minus any sample and is processed through the entire extraction quantification amplification and electrophoretic typing procedures If more than one type of extraction procedure is employed with different extraction reagents then a reagent blank must be set up for each type of extraction or group of extraction reagents used Twenty microliters of the reagent blank should be amplified The reagent blank must be run in at least one of the PCR systems to check for contamination of the reagents with genomic DNA Extraction Allelic Control The purpose of this control is to insure that the extraction and typing procedure worked properly The control may be either a bloodstain or other biological material from a previously characterized source that suitab
158. d Reynolds R AmpliType PM and HLA DQa Typing from Pap Smear Semen Smear and Post coital Slides Journal of Forensic Sciences 40 1995 266 9 Fildes and Reynolds 1995 Consistency and Reproducibility of AmpliType PM Results Between Seven Laboratories Field Trial Results Journal of Forensic Sciences 40 279 86 Perkin Elmer AmpliType User Guide Version 2 1990 Gyllensten U B and Erlich H A Generation of Single Stranded DNA by the Polymerase Chain Reaction and Its Application to Direct Sequencing of the HLA DQa Locus Proceedings of the National Academy of Sciences USA 85 1988 7652 6 Yamamoto T Davis C G Brown M S Schneider W J Casey M L Goldstein J L and Russell D W The Human LDL Receptor A Cysteine rich Protein with Multiple Alu Sequences in its mRNA 39 1984 27 38 Kudo Shinichi and Fukuda Minoru Structural Organization of Glycophorin A and B Genes Glycophorin B Gene Evolved by Homologous Recombination at Alu Repeat Sequences Proceedings of the National Academy of Sciences USA 86 1989 4619 23 Slightom J L Blechl A E and Smithies O Human Fetal Gy and Ay globin Genes Complete Nucleotide Sequences Suggest That DNA Can Be Exchanged Between These Duplicated Genes Cell 21 1980 627 38 Horn G T Richards B Merrill J J and Klinger K W Characterization and Rapid Diagnostic Analysis of DNA Polymorphisms Closely Linked to the Cystic Fibro
159. d the work area e Absorbent pads are placed on bench tops and are changed as necessary Removal of equipment supplies and waste material is accomplished by one of the following methods 1 ultraviolet light exposure for a minimum of 20 minutes per surface area or 2 double bagged in autoclave bags and taken directly to the autoclave STERILIZATION Autoclaves are used for treatment of wastes prior to disposal such as PCR products and blood tubes from database samples and for the sterilization of reagents and materials Whenever possible autoclaved materials are sterilized in the area where they are used or produced CLEANING AND DECONTAMINATION If a lab bench is contaminated the absorbent pads should be folded inward on themselves and placed into an autoclave bag This autoclave bag should be double bagged and taken directly to the autoclave The lab bench should then be washed with a 10 bleach solution followed with a 95 ethanol rinse New absorbent pads should be placed on the surface If a thermal cycler becomes contaminated all of the tubes should be removed and discarded If using the 480 Thermal Cycler the oil should be removed from all the wells using cotton tipped applicators Completely clean the block and affected surfaces with a 10 bleach solution Ultraviolet light treat all affected surface areas for at least 20 minutes per surface area using an UV light source Rinse with 95 ethanol If other pieces of equi
160. d when necessary to assess the background contribution potential of evidence stain substrates These specimens are processed with evidence samples and are required when there is reasonable expectation of detectable background DNA in an evidence specimen A negative amplification blank is required with each set of amplifications This control demonstrates the integrity of the amplification kit components as well as serving as a check for contamination during PCR reaction setup It consists of a PCR cocktail to which sterile deionized water or sterile TE Buffer instead of DNA is added A positive DNA control is required with each set of amplifications to test for success of amplification and to demonstrate that all aspects of the PCR reaction process are performing as expected in terms of specificity and sensitivity The manufacturer supplies this control as a kit component or a NIST traceable standard that is extracted at the same time as the samples may be used CASEWORK A tissue extraction control is a blood sample that is a NIST traceable standard with known DNA profile It is extracted quantitated amplified and typed with each set of blood buccal and tissue samples The tissue extraction control may also be used to verify commercial kits extraction reagents thermal cyclers and typing instruments CASEWORK A differential extraction control is a sperm non sperm mix sample that is a NIST traceable standard with a known DNA profile It is
161. dded if analyzed ldentifiler Interpretation of the results will follow current CB Forensic Laboratory DNA Analysis SOP protocol for casework The analyst will transmit the results of the proficiency test to the Quality Assurance Manager QAM or designee The analyst will maintain the original data calculations and other information in a notebook with a copy of the results to be given to the QAM or designee All guidelines written in the CB Forensic Laboratory Quality Assurance Manual are followed and the test handled as if it were evidence A memo from the QAM or designee is given to the analysts regarding the results of their testing Any discrepancies within a proficiency test are handled according to the CB Forensic Laboratory Quality Assurance Manual When the consensus report is issued from the supplier of the proficiency test the Technical Leader will review the report and then compare their results to those of each analyst who participated in the test After the comparison the Technical Leader will initial the analyst s proficiency test showing compliance to the consensus report A memo which can be in electronic form will be sent to the QAM from the Technical Leader that addresses the evaluation of all participants in the proficiency test Any discrepancies between the analyst and the consensus will be handled according to the CBI Forensic Laboratory Quality Assurance Manual TESTING REQUIREMENT PRIOR TO CASEWORK Following the
162. door in a secure building The following security procedures have been implemented A The CODIS workstation must be logged on and off each time CODIS is used B Only FBl approved personnel have User Id names and passwords for CODIS Persons other than those approved by the FBI are not authorized to have access to the CODIS database C The CJIS WAN router for CBI Denver laboratory the SDIS router is located in the locked central computer room D ALL CODIS users are responsible for the security of the software TAPE BACKUP PROCEDURES The CODIS servers are set up to do a nightly tape backup Both the Local CODIS Administrators and the State CODIS Administrator will do two full tape backups once a month One will be stored in each laboratory The second will be stored off site FILE STORAGE A Copies of DNA case reports and the associated tables will be stored in the DNA laboratory The DNA laboratory is secured behind a keypad door within a secure building B The file containing the identification information for the Convicted Offender Database is kept on a stand alone computer at the Denver laboratory Access to this computer is limited to the persons involved with the Convicted Offender Database Backups of the personal information are performed whenever entries are made Contents of this file will be compared to the specimen numbers entered into CODIS on a yearly basis for quality assurance purposes Page 175 of 255 CBI DNA A
163. e PREPARATION OF REAGENTS Before starting UV treat all equipment and glassware 20 minutes per surface area or autoclave A BSA 8 ug ul BOVINE SERUM ALBUMIN Add 8 g BSA Sigma A2153 per every 1 L 0 8 g per every 100 ml autoclaved ultrapure water Mix thoroughly Sterile filter through a Nalgene 0 2 or 0 45 micron filter unit Aliquot into sterile 15 ml tubes Aliquot from 15 ml tube into sterile 1 5 ml Sarstedt tubes as needed Store all tubes at 20 C Page 26 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 CHELEX SOLUTION 20 and 5 w v 100 ml 1 20 w v Chelex Solution Add 20 Chelex to 100 ml sterile ultrapure Aliquot into sterile 1 5 ml tubes Check that pH is greater than 9 Store at room temperature protected from light by covering the test tube rack with foil 2 5 w v Chelex Solution Add 5 g Chelex to 100 ml sterile ultrapure Aliquot into sterile 1 5 tubes Check that pH is greater than 9 Store at room temperature protected from light by covering the test tube rack with foil Needed for sperm containing samples only When pipetting Chelex Stock Solutions the resin beads must be distributed evenly in solution this can be achieved by gentle mixing with a stir bar in a beaker Also the pipette tip used must have a relatively large bore 1ml tips are adequate 10X CITRATE BUFFER 1M SODIUM C
164. e and 7 ul of 1M DTT Mix gently Incubate at 37 C for 30 minutes to 1 hour Vortex epithelial and sperm cell samples at high speed for 5 to 10 seconds Spin in a microcentrifuge for 10 to 20 seconds at 10 000 to 15 000 x g Incubate the samples in a boiling water bath for 8 minutes Vortex at high speed for 5 to 10 seconds Spin in a microcentrifuge for 2 to 3 minutes at 10 000 to 15 000 x g The samples are now ready for the PCR quantification and amplification process lt x ESS CHM D Store the remainder of the supernatant at either 2 to 8 C or frozen To re use repeat Steps V through X Page 35 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 SEMEN WHOLE SEMEN A Add 3 ul of whole semen to 200 ul of 5 Chelex in a 1 5 ml microcentrifuge tube B Add 2 ul of 10 mg ml Proteinase and 7 ul of 1 M DTT Mix gently C Incubate at 56 C for 30 to 60 minutes Vortex at high speed 5 to 10 seconds D Spin in a microcentrifuge for 10 to 20 seconds at 10 000 to 15 000 x g E Follow protocol for Whole Blood Blood Stains beginning with Step H SEMEN STAINS A Dissect stain material Use a clean cutting surface for each different sample B Pipet 1ml of PBS into a sterile 1 5 ml microcentrifuge tube Add fabric cutting C Incubate at room temperature for 30 minutes D Vortex the tube at high speed for 1 minute Remove the fabric with a toothpick or pipette tip
165. e NaH2PO 4eH20 Adjust the pH to 7 4 0 2 with 10N NaOH about 10 ml Adjust the final volume to 1 liter with ultrapure water and mix thoroughly Autoclave and store at room temperature STERILE SALINE Dissolve 8 5 NaCl in 1000 ml sterile ultrapure water Autoclave Page 30 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 BB TBE BUFFER 89mM TRIS BORATE 2 0mM EDTA pH 8 3 1 10X TBE CONCENTRATED STOCK 0 89M TRIS BORATE 0 020M EDTA 11 To 40 ml 0 5M EDTA pH 8 0 add approximately 800 ml ultrapure water Add 108 g Tris base and 55 g Boric Acid to the diluted EDTA solution Stir vigorously on a magnetic stirrer Adjust volume to 1L with ultrapure water Filter using a 0 2 or 0 45 micron Nalgene filter unit Store at room temperature 2 1X TBE WORKING STOCK Immediately before use dilute one part 10X Concentrated TBE Stock with nine parts ultrapure water CC TE BUFFER 10mM Tris HCI 0 1mM EDTA pH 8 0 1L Mix together 10 ml 1M Tris HCl pH 8 0 0 2 ml 0 5M EDTA and 990 ml ultrapure water Sterilize by autoclaving Store at room temperature DD TRIS EDTA NaCI 10mM Tris HCI 100mM NaCl 1mM EDTA pH 8 0 100 ml Add 1ml 1M Tris HCl to approximately 75 ml ultrapure water To this solution add 0 584 g NaCl and 200 ul 0 5M EDTA Stir until dissolved Adjust the pH to 8 0 with 1N NaOH and bring to a final volume 100 ml with ultrapure water Au
166. e Add the following reagents in the order listed to a glass flask and mix thoroughly by swirling Do NOT vortex To 30 ml Citrate Buffer add 1 5 Chromogen TMB Solution and 30 ul 3 H202 2 Add Color Development Solution to the membrane in the tray Cover the tray with the lid to protect the membrane from strong light 3 Shake at room temperature on an orbital shaker 50 to 60 rpm for 20 to 30 minutes until all of the DNA Standards are visible Remove tray from shaker pour off liquid Stop the color development by washing in deionized 100 ml Shake for 5 to 10 minutes 50 to 60 rpm with the lid on the tray Repeat for a total of three washes 6 Photograph the membrane when it is wet Saran Wrap may be placed over the membrane during photography to prevent it from drying out a Place the wet membrane on a flat non absorbent surface Keep the membrane wet throughout the photographic procedure Minimize exposure to strong light b Use a Polaroid camera with Polapan 400 or Type 52 black and white film or Type 59 or 559 color film or other photographic recording device Follow exposure development instructions NOTE For black and white photography an orange filter Wratten 22 or 23A will enhance contrast d Following photography the membrane may be air dried on any hard non absorbent surface Protect from light and oxidizing agents e g acid treated paper bleach and nitric acid The blue
167. e CgHgO7 e H20 Bring up to 2 liter with ultrapure water mix and autoclave Date Prepared Amount 5 2 H20 lot CgHg07 H20 lot pH Preparer 10CITBUF Page 220 of 255 Appendix N DNA Stock Solutions Log 10X Citrate Buffer CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 APPENDIX O DNA Stock SoLurioNs Loc 0 5M EDTA PH 8 0 MODEL Solution Preparation Slowly add 186 1 g disodium ethylenediaminetetraacetic acid dihydrate NazEDTA 2H2O to 800 ml ultrapure water Stir vigorously on a magnetic stirrer Adjust the pH to 8 by adding NaOH pellets approximately 20 g Check pH and use 10 N NaOH if needed for small pH adjustments Note EDTA will not go into solution without adjusting the pH Bring to 1 liter with ultrapure water Sterilize by autoclaving Store at room temperature Date Prepared Amount NajEDTA 2 H20 lot NaOH pellets lot 10 N NaOH lot pH Preparer Page 221 of 255 Appendix O DNA Stock Solutions Log 0 5M EDTA pH 8 0 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 EDTA2 Page 222 of 255 Appendix O DNA Stock Solutions Log 0 5M EDTA pH 8 0 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 APPE
168. e in 80 ml ultrapure water Adjust pH to 5 2 adding glacial acetic acid approximately 2 ml Adjust the final volume to 100 ml Sterilize by autoclaving Store at room temperature Date Prepared Amount Sodium Acetate lot Glacial Acetic lot Preparer NAOAC Page 225 of 255 Appendix R DNA Stock Solutions Log 1M Sodium Acetate pH 5 2 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 APPENDIX S DNA STOCK SOLUTIONS Loc 20 w v SoDIUM DODECYL SULFATE MODEL SDS 11 Solution Preparation Warning SDS is an irritant Avoid skin contact and inhalation Slowly dissolve 200 g electrophoresis grade ultrapure SDS in 800 ml ultrapure May need to warm solution i e 50 C incubator to dissolve the SDS Adjust to 1 liter using ultrapure water Mix thoroughly Amount SDS Date Prepared Lot Preparer 20 SDS2 Page 226 of 255 Appendix S DNA Stock Solutions Log 20 w v Sodium Dodecyl Sulfate CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 APPENDIX T DNA STOCK SOLUTIONS Loc 20X SSPE BurrER MODEL 3 6M NACL 200MM NAH2PO eH20 20MM EDTA 7 4 2L Solution Preparation In 1600 ultrapure water dissolve 14 8 Na2EDTAe2H20 Adjust pH
169. e DNA extraction PCR setup room of the laboratory Setup of PCR reactions must be separated by time from other uses of the PCR workstation This area is also used for dispensing PCR reaction mix aliquots into the PCR reaction tubes The following special precautions apply while setting up PCR reactions FTA samples exempt A The PCR workstation must be irradiated with ultraviolet light for 20 30 minutes before setting up PCR reactions Pipettors vortexors pipette tips sampling instruments labeling instruments decappers timers and microtube racks remain in the PCR workstation during ultraviolet irradiation B After addition of the PCR reaction mix to the tubes all tubes are capped After addition of the extracted DNA sample to the appropriate PCR reaction tube the tube is recapped before proceeding to the next DNA sample C Gloves are changed as necessary to avoid contaminating other specimens D Kit reagents are handled and stored such that they are not exposed to extraneous DNA Mineral oil is not to be exposed to be ultraviolet light as this can cause the oil to inhibit PCR Microtube racks that are used for carrying the completed PCR reaction mix tubes to the thermal cycler in the PCR amplification and typing room s must be exposed to a minimum of 20 minutes of ultraviolet light per surface area before they are returned to the PCR workstation in the DNA extraction PCR setup room PCR AMPLIFICATION AND TYPING AREA Acces
170. e inside face down at the bottom end of the notched plate B Prepare pre packaged Long Ranger gel solution by following the instructions on the gel pack Two gels can be poured from one Singel pack C Make 1500 ml 1X TBE by mixing 150 ml 10X TBE with 1350 ml ultrapure water D Once the gel solution is prepared squeeze it into a glass beaker Then draw the solution up into a 60 ml syringe Pour the gel by applying the solution between the plates and gradually sliding the top plate along the bottom plate until it reaches the end Make sure that the two plates are even and that the spacers are positioned properly Insert the casting comb in the well region Place clamps along the sides of the glass plates and along the comb DO NOT place clamps along the bottom of the glass plates A second gel may be poured at this time E After 15 minutes wrap the bottom end of the gel with 1X TBE wetted Kimwipes and plastic wrap Cover the remaining 1X TBE with plastic wrap For overnight storage Page 134 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 wrap the gel completely with plastic wrap after it has polymerized for a minimum of 1 hour MACINTOSH PLATFORM PROCEDURE WARNING CHEMICAL HAZARD Formamide is an irritant and teratogen Avoid skin contact and inhalation Use in a well ventilated area Wear lab coat and gloves when handling A Turn on the ABI PRISM 377 DNA Sequenc
171. e intensity of the DNA Standards 2 Divide this quantity by the volume of DNA test sample added to the Spotting Solution typically 5 DNA test sample is added to 150 ul Spotting Solution This calculation gives DNA concentration in ng ul PERFORMANCE CHARACTERISTICS The QuantiBlot Human DNA Quantitation Kit will be able to detect and quantitate 0 15 to 10 ng of human DNA per test sample TROUBLESHOOTING OBSERVATION POSSIBLE CAUSE RECOMMENDED ACTION 1 No signal low Use of a membrane other Use Biodyne B nylon sensitivity 0 15 ng DNA than Biodyne B membrane Do not use Standard not visible membranes that have a neutral charge Incorrect NaOH or EDTA Prepare Spotting Solution concentrations in Spotting correctly Solution Page 62 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 OBSERVATION 2 Areas of low sensitivity across the membrane JUNE 2002 POSSIBLE CAUSE Water bath temperature too high DNA Probe was not added at hybridization step Enzyme conjugate was not added Hydrogen inactive peroxide was Presence of MgCl in the DNA sample Membrane slipped up onto the side of the Hybridization Tray during Hybridization or Stringent Wash steps Membrane dried out sig nificantly at some point in the protocol Page 63 of 255 CBI DNA Analysis SOP Vers 2 1 RECOMMENDED ACTION Water bath temperature should be 50 C 1 C
172. e law enforcement agencies can share DNA information obtained from convicted offenders and forensic evidence This system allows agencies to cross reference case evidence profiles with those of other agencies and to generate investigative leads by comparing forensic DNA profiles with profiles from convicted offenders All criminal justice agencies are eligible to participate in CODIS if they perform DNA analysis using specific loci and adhere to the NDIS Standards for Acceptance of DNA Data CODIS accepts DNA profiles derived from either RFLP analysis or PCR analysis Currently there are three levels of CODIS Local DNA Index System LDIS State DNA Index System SDIS and National DNA Index System NDIS Each local system contains databases consisting of DNA profiles analyzed at that laboratory The local systems all upload the DNA profiles from their databases to the designated state system each state has one designated SDIS location Thus the state index consists of profiles collected from all participating LDIS laboratories The State Administrator uploads all appropriate profiles contained in SDIS to the national system The Federal Bureau of Investigation FBI maintains the national system CBI has three local systems located in the Denver Pueblo and Montrose laboratories CBI Denver laboratory is also designated as the SDIS location for Colorado CBI maintains two CODIS databases or indexes the Convicted Offender Database and the Fo
173. e technical review process for database profiles involves merging the typing results obtained by two analysts into one table The profiles are then compared and any discrepancies in results are resolved prior to CODIS entry There are two methods for merging Genotyper allelic files A For the Macintosh platform the Genotyper allelic tables from both analysts are imported into Microsoft Excel merged together and sorted so that corresponding samples are side by side The merged tables are printed and the profiles from the Page 156 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 two analysts are visually compared to insure that all alleles match Both analysts must initial the table indicating that they are in agreement For the Windows NT platform CODIS tables are created in Genotyper In the dye lanes window remove all controls RB NAB and positive controls and ladders Select samples to be removed by highlighting them then select Clear under Edit Create a CODIS table by executing the Make CODIS Table macro Export the CODIS table to the folder saving the table CODISGTdateanalD PP CO TABLE The reviewing analyst follows the same steps but includes their initials at the end of the reviewed folder i e CODISGTdateanalD PP CO revTABLE The two tables are merged together in the NT CMF conversion software which also compares the results and creates a report
174. e tube containing the washed paper punch sample then add PCR amplification mix directly to the punch containing the purified immobilized DNA Page 54 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 This Page Intentionally Left Blank Page 55 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 QUANTIBLOT A Reagent Preparation 1 Preparation of reagents supplied Chromogen TMB Solution Bring the Chromogen TMB TMB to room temperature 15 to 30 C Before opening the bottle tap it on the lab bench to shake the TMB to the bottom of the bottle Remove the stopper carefully to prevent loss of the powder Slowly add 30 ml of room temperature reagent grade 100 ethanol to the bottle Do NOT use ethanol that has been stored in a metal container Do NOT use 95 ethanol or other alcohols Recap the bottle Seal the stopper with Parafilm Shake in an upright position on an orbital shaker for 30 minutes or until completely dissolved Store in bottle at 2 to 8 C and protect from rust Under these conditions the Chromogen Solution is stable for six months after preparation 2 Human DNA Standards Prepare a two fold serial dilution of the DNA Standard A provided in Kit in TE Buffer as follows a Label seven 0 5 ml DNA RNA free microcentrifuge tubes A through G b Vortex the DNA Standard A to mix it thoroughly
175. ecords The State CODIS Administrator shall notify the NDIS Custodian of the expungement and forward the appropriate paperwork NDIS QUALITY ASSURANCE QUALITY CONTROL STANDARDS PROFICIENCY TESTING A All qualified DNA technicians and analysts conducting casework will participate in two external proficiency tests per year These tests will be conducted on a semiannual basis at regular intervals not to exceed 183 days as per the FBI Quality Assurance Standards Any questions problems related to a proficiency test will be handled as detailed in the Forensic Laboratory Quality Manual All QA QC guidelines outlined in the CBI Forensic Laboratory DNA Analysis SOP and the CBI Forensic Laboratory Quality Manual will be followed Proficiency test documentation will be provided as specified by current NDIS procedures Page 174 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 AUDITS A The DNA sections will be audited once a year There shall be not less than six months or more than eighteen months between audits External audits will be performed during alternate years B Audit documentation will be provided as specified by current NDIS procedures SYSTEMS OPERATIONS CODIS SECURITY The CODIS servers are located in locked rooms with limited access Individual workstations are located in analysts offices The offices are within the CBI laboratories behind a keypad
176. ele calls below 50 RFUs E Highlight to analyze all four colors F Click Analyze Page 146 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 G Review the samples individually in Analysis Control Note Once the optimal analysis parameters have been established the Auto Analysis function may be used for all runs 1 Examine the negative amplification blank and reagent blank for the presence of any contaminating peaks Examine the positive controls to insure that all peaks are present and sized Evaluate the samples with the following parameters a Peak shape and height b Matrix quality Peak profile examine for artifactual peaks d Presence of any spurious peaks due to leakage or spillover H Low level samples may be re analyzed with a lower down to 50 RFU peak height threshold Some samples may require the approval of the Technical Leader l After the review is complete save any changes GENESCAN ANALYSIS USING NT SOFTWARE A Begin by copying the run folders of interest onto your workstation s desktop from the shared folder of the server that ties the 3100 to the workstations B Open GeneScan and create a new project by adding sample files of interest from the desktop Save project as GSdateanalD PP CO C Review a representative number of samples raw data to determine the Analysis Range D Within the Analysis Control Window select all colors pres
177. ency case number date the blood was drawn and the initials of the person collecting the sample B The protective packaging containing the buccal sample will be labeled with the offender s name first and last agency case number and the date the sample was collected Storage Before Submitting Sample to A Blood samples are required to be refrigerated in order to keep the samples cold at all times and submitted to the CBI Denver Laboratory within one week of collection Blood should not have been frozen at any time B The buccal sample should be thoroughly dried and placed in a protective package kept at room temperature Submission The CBI Database Submission form must accompany a sample The form will be kept separate from the blood tube or buccal sample and its sealed container CBI HANDLING OF DATABASE SAMPLES Submission A At the time of sample submission to the Denver Laboratory CBI personnel will sign the chain of custody and assign a CBI case number Database personnel will then enter the submission information into a catalog database assign each sample a confidential PCR number and dispense the necessary paperwork The samples will be stored appropriately Page 25 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 If the sample does not meet the requirements described in the Submission section CBI personnel will use discretion as t
178. ended that the positive control negative control and reagent blank be injected twice on each run E Bracket each run of samples with the appropriate ladder Profiler Plus or Cofiler to insure at least one comparable ladder is available for each sample This may require that the ladder be injected several times throughout the run Page 108 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 NOTE It may be beneficial to prepare two ladder tubes for each run NOTE The Matrix column should indicate either lt none gt or the name of the matrix made j Fill in the name and click the Run button NOTE If necessary additional injections can be added to the analysis run during the run 310 analysis run complete 1 Once the run is completed save the Injection List and the Analysis Run 2 Save the Analysis folder as follows RunFolder date analyst s initialsPP and or CO e g run folder 01 22 00xyzPP or run folder 012200xyzPP 3 Once saved transfer the folder along with the notes and other necessary data to an analysis computer 4 From the Manual Control select the Present Tray from the pop up menu Remove the tray and from the Manual Control select the Return Tray from the pop up menu then click Execute 6 If completed with all runs the instrument will be powered down by a discarding buffer vials b cleaning the pump block placing the c
179. ent for analysis Insure that the appropriate dye color is marked as the size standard a diamond symbol should be present in the orange boxes for ID or red boxes for PP CO E Select the Define New option from the Size Standard pop up menu next to the sample you wish to use to define the new size standard Define the standard peaks as 75 100 139 150 160 200 300 340 350 400 and 450 Note For capillary electrophoresis the 250 is labeled as 0 F Insure that the Analysis Parameters are as follows 1 Analysis Range Select a range of data points incorporating both the 75 and 400 bp size standards 2 Data Processing Light Smooth Option Page 147 of 255 CBI DNA Analysis SOP Vers 2 1 12 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 Peak Detection B 150 G 150 Y 150 R 150 O 150 Min Peak Half Width 2 pts Polynomial Degree 3 Peak Window Size 19 pts Slope Threshold for Peak start 0 0 Slope Threshold for Peak end 0 0 Size Call Range Min 75 Max 400 Size Calling Local Southern Method Baseline Window Size 251 pts Auto Analysis Size Standard None selected Highlight all colors present to analyze by clicking on the top left box of the grid Click Analyze View results In the Analysis Control window Negative controls Review the negative controls for the presence of any contaminating peaks Page 148 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATO
180. er B Start the computer If the computer was left on from a previous run restart it Launch the ABI 377XL Collection software if it is not already open C Remove deionized formamide from the freezer to thaw D Prepare a sample sheet this can be done in the DNA extraction PCR setup room and transferred over the network The Macintosh should be restarted after the transfer 1 Choose NEW from the File menu in 377XL Collection software 2 Click on the GeneScan Sample Sheet icon 3 Fill in the sample name column first The sample name is the PCR number followed in sequence by the case number i e PCR ZYR H When complete highlight the Sample Name column by clicking once in the gray bar at the top and select Copy from the Edit menu 4 Highlight the Sample Info column and select Paste from the Edit menu Note Under the Sample Info column check that each sample has a unique sample number Also check that each lane that contains an allelic ladder has the word ladder in the Sample Info column this is necessary for automated allele calling with Genotyper v 2 0 or higher 5 Check that there is a diamond next to the red box in the Std column for each sample 6 Make sure there is an X in each box in the PRES column for the dye colors to indicate which dye colors are to be collected Check that the Std column designates red Under the Sample Info column check that each sample has a unique sample numb
181. er Also check that each lane that contains an allelic ladder has the word ladder in the Sample Info column this is necessary for automated allele calling with Genotyper v 2 0 or higher 9 Select Save As from the File menu and label the sample sheet Page 135 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 Prepare notes sheet Include the following information as minimum Analyst e Date Instrument Long Ranger lot e 10X TBE lot e Formamide lot Amplification date Sample dilutions Control dilutions Load volume F Prepare the samples for analysis 1 Remove the GeneScan ROX 500XL ROX 500XL internal lane standard and allelic ladders from the refrigerator Vortex and spin 2 Prepare formamide loading solution FLS by combining 500 ul of formamide with 100 ul blue dextran 75 mg ml 25mM EDTA Mix by vortexing 3 Prepare FLS ROX by adding ROX 500XL to the FLS at a concentration necessary to obtain an RFU value of 50 to 1000 in the reagent blank and negative amplification blank The concentration of ROX 500XL can vary depending on lot and instrument sensitivity Make a sufficient quantity for all samples Mix the tube by vortexing Place the appropriate number of 0 5 ml tubes in a tube rack and label appropriately 6 Add to each tube the required amount of FLS ROX mixture then cap all tubes T Add the required am
182. ered by CODIS software at a single instant in time Hits may occur at any level in the CODIS hierarchy LDIS SDIS or NDIS There are two categories of hits A Forensic Hit FH occurs when two or more forensic samples are linked at LDIS SDIS or NDIS Forensic hits are sometimes called case to case hits Page 185 of 255 Appendix B Terms and Definitions for CODIS DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 An Offender Hit OH occurs when one or more forensic samples are linked to a convicted offender sample at SDIS Offender hits are sometimes called case to offender hits Investigation Aided This occurs when a CODIS hit provides information that assists a criminal investigation Keyboard Search A manual search of CODIS initiated by a CODIS user LDAS Local DNA Analysis System The data entry part of CODIS Profiles and the accompanying information can be entered into LDAS either by using the computer keyboard for hand entry or using CODIS Import for electronic transfer LDIS Local DNA Index System The locally administered centralized system of DNA identification records containing forensic unknowns and convicted offender profiles Match Report After CODIS determines that two or more DNA profiles match this electronic report is generated by CODIS and automatically distributed to the laboratories responsible for matching profiles Match stringency
183. ers 2 1 06 13 02 4 43 PM CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 ALLELE FREQUENCIES CONTINUED FGA African Southwest Southeast Allele American mE N 180 N 198 N 203 N 191 lt 18 0 0139 0 0128 0 0123 0 0131 18 0 0139 0 0306 0 0123 0 0131 18 2 0 0139 0 0128 0 0123 0 0131 19 0 0528 0 0561 0 0788 0 0838 19 2 0 0139 0 0128 0 0123 0 0131 20 0 0722 0 1454 0 0714 0 1178 20 2 0 0139 0 0128 0 0123 0 0131 21 0 1250 0 1735 0 1305 0 1361 21 2 0 0139 0 0128 0 0123 0 0131 22 0 2250 0 1888 0 1773 0 1492 22 2 0 0139 0 0128 0 0123 0 0131 22 3 0 0139 0 0128 0 0123 0 0131 23 0 1250 0 1582 0 1404 0 1492 23 2 0 0139 0 0128 0 0123 0 0131 24 0 1861 0 1378 0 1256 0 1466 24 2 0 0139 0 0128 0 0123 0 0131 24 3 0 0139 0 0128 0 0123 0 0131 25 0 1000 0 0689 0 1379 0 1126 26 0 0361 0 0179 0 0837 0 0471 26 2 0 0139 0 0128 0 0123 0 0131 27 0 0222 0 0128 0 0320 0 0262 28 0 0167 0 0128 0 0123 0 0131 29 0 0139 0 0128 0 0123 0 0131 30 0 0139 0 0128 0 0123 0 0131 Page 199 of 255 Appendix D Allele Frequencies CBI DNA Analysis SOP Vers 2 1 06 13 02 4 43 PM CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 ALLELE FREQUENCIES CONTINUED Appendix D Allele Frequencies CBI DNA Analysis SOP
184. es can be spotted on the membrane for a total of up to 48 samples per hybridization reaction DNA Calibrators are provided as an internal control for DNA Standard performance C Slot Blotting Immobilization of DNA NOTE Wear clean disposable laboratory gloves while preparing samples Follow safety recommendations provided by manufacturer for handling chemicals Comply with any and all laws regulations or orders with respect to the disposal of any hazardous or toxic chemical material substance or waste Page 57 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 Before beginning this section assemble the required reagents supplies and equipment as follows DNA Standards A through G DNA Calibrator 1 provided in Kit DNA Calibrator 2 provided in Kit Slot Blot Apparatus Spotting Solution Pre Wetting Solution Biodyne B nylon membrane 1 Determine the number of samples to be analyzed including the seven Human DNA Standards A through G the DNA Calibrators 1 and 2 provided in Kit and the one blank Spotting Solution only Aliquot 150 ul Spotting Solution into a new 0 5 ml GeneAmp PCR Reaction Tube for each sample 2 Label seven of the tubes containing 150 ul Spotting Solution as follows A B C D E F and G Label two other tubes containing 150 ul Spotting Solution as DNA Calibrator 1 and DNA Calibrator 2 3 Vortex the seven DNA standards and the t
185. esent A The allele frequency for each allele greater than or equal to 50 RFU in the mixed DNA profile for an individual locus is determined B The individual fragment frequencies are summed then this value is squared B4 Pe tage where n equals the number of alleles present and Piocus is the probability of an unrelated individual in the population being a contributor to that mixture at that locus C Multiply each value of to generate the probability of an unrelated individual in the population being a contributor to that mixture for all loci examined 1 X Procus 2 X Procus n Puis Where equals the number of loci analyzed and present the mixture Page 118 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 D Calculate the percent of the population that can be excluded as a possible donor to the mixture of DNA in the sample as follows 100 1 CRIMINAL PATERNITY CASES In rare cases it may be necessary to evaluate by genetic testing a parentage of a biological substance The Colorado Bureau of Investigation will participate in these types of cases in a limited capacity The genetic testing of the material will be performed according to the current Colorado Bureau of Investigation Standard Operating Procedure The alleles found in the child s sample are referred to as obligate alleles that is the child must have inhe
186. eservoir to the red line with 1X Genetic Analyzer Buffer with EDTA and replace the reservoir on the pump block 3 Fill a 4 ml glass buffer vial to the full line with 1X Genetic Analyzer Buffer with EDTA Insert the plastic vial lid with attached septum into the glass vial Place this buffer vial in position 1 on the Autosampler This is the cathode buffer 4 Fill a second 4 ml glass buffer vial to the fill line with filter purified water Insert the plastic vial lid with attached septum into the glass vial Place this buffer vial in position 2 of the Autosampler 5 Fill a 1 5 ml microcentrifuge tube cut off the lid with filter purified water and place into position 3 on the Autosampler Do not use a screw top tube NOTE To avoid electrical arcing it is imperative that all surfaces of the buffer vials and the microcentrifuge tube be clean and dry N Calibrating the Autosampler NOTE Autosampler calibration should be performed whenever the capillary or electrode is changed or whenever the position of the electrode is altered 1 Choose Autosampler Calibration from the Instrument menu 2 Select Start remove Autosampler tray if necessary and select Resume 3 Follow the instructions on the screen to set the calibration point Click the direction arrows to align the front calibration point of the Autosampler with Page 104 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2
187. essary proceed to Step G 1 Unscrew the electrode thumbscrew located to the right of the detector door and just below the heat plate 2 Insert the long end of the new platinum electrode into the electrode hole of the thumbscrew 3 Insert the hooked end of the electrode into the outer hole 4 Replace the electrode back on the instrument Under the Manual Control window select Autosampler Home Z Axis from the Function pop up menu click Execute 6 Use flush cutting wire cutters with the flat cutting surface facing the top of the instrument to cut off a small piece of the electrode so that it is flush with the lower surface of the stripper plate Page 101 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 Removing and cleaning the syringe 1 If the syringe drive is not in the home position select Syringe Home from the Function pop up menu of the Manual Control window 2 Move the drive toggle to the left position to allow for removal of the syringe from the pump block 3 Unscrew the syringe to remove it from the pump block 4 Clean and rinse the 1 0 ml syringe with warm tap water if necessary then with filter purified water Remove all excess water from the inside and outside of the syringe using compressed air Priming the syringe 1 Draw up 1 0 of filter purified water and then expel 2 Draw up approximately 0 2 ml of room temperature POP 4 into the
188. essional judgment and expertise The following objective criteria are to be used by the analysts to aid in their interpretation of the data presented them These criteria are based upon validation studies literature and experience They are not a set of hard and fast rules but rather a means to establish a general framework and outline minimum standards to insure that conclusions are scientifically supported by the analytical data including that obtained from appropriate standards and controls interpretations are made as objectively as possible and interpretations are consistent from analyst to analyst EVALUATION OF STR DATA The goal of the evaluation and interpretation of the STR data is to correctly determine the DNA profiles from database samples for inclusion into CODIS The following should be applied to aid in that interpretation A peak is defined as a triangular section of the electropherogram that results from the sum of input signals and as such shows Gaussian distribution The internal size standard ROX or LIZ must have the correct sizes assigned to the peaks used for sizing The 75 350 bp peaks must be captured when using the Cofiler reagent kit The 400 bp peak must be captured when using the Profiler Plus or Identifiler reagent kits The ladder used by Genotyper to call the alleles of the samples analyzed must be correctly labeled with the correct allele designations Genotypes are determined from the dia
189. eviewer Technical Leader and or Agent In Charge in order to achieve a reportable opinion SiNGLE SOURCE CONTRIBUTORS A sample can be considered to have originated from a single source if A Only one or two alleles are present at all loci examined although three peak patterns can occur and or B The peak height ratios of the heterozygotes fall within expected values Population statistics will be calculated for probative single source samples when allele peaks represented are greater than or equal to 150 RFU Locus Evaluation Both peaks of a heterozygote must be represented and be greater than or equal to 150 RFU A locus can be determined to be inconclusive and a value for the frequency of that locus assigned the value of one if it is below 149 RFU If an exclusion can be made with peaks below a value of 149 RFU then the allele can be used in evaluation In the event that all loci are below 150 RFU the sample can be concentrated and re evaluated if sample remains If an exclusion can be made with this information it must be used MULTIPLE SOURCE CONTRIBUTORS A sample can be considered to have originated from multiple two or more sources if A More than two alleles are present at two or more loci although three peak patterns can occur from a single source sample and or B The peak height ratios for heterozygotes fall outside the expected values when all loci are considered Note Alleles with peak heights between 50 and
190. extracted quantitated amplified and typed with each set of sperm samples The differential extraction control may also be used to verify commercial kits extraction reagents thermal cyclers and typing instruments DATABASE The Internal DNA control is a blood sample that has a known DNA profile and is a NIST traceable standard This control is also used to verify commercial kits extraction reagents thermal cyclers and typing instruments Human DNA quantitation by QuantiBlot amp is required on all samples and tissue extraction controls that have been extracted for casework analysis Reverse dot blots are examined for the presence of the control C and S dot for interpretation A second analyst performs a technical review on all typing results Page 18 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 EQUIPMENT CALIBRATION AND MAINTENANCE The following equipment controls are employed in the PCR procedures All procedures mandated by the CB Forensic Laboratory Quality Assurance Manual are followed THERMAL CYCLER A The Temperature Calibration Verification Test of the thermal cycler is performed biannually using a calibrated temperature verification kit B The Temperature Uniformity Test of the thermal cycler is performed biannually using a Calibrated temperature verification kit C The Perkin Elmer 480 thermal cycler sample block is cleaned after each use with cotton swab
191. for sale In this Laboratory their performance as well as the performance of in house reagents is monitored with each extraction amplification and typing run by the use of a known reagent blank positive control and negative amplification blank which are outlined below The integrity of the components of each Amplitype amp AmpF STR Profiler Plus AmpF STR Cofiler kit and AmpF STR Identifiler kits as well as the in house prepared reagents is maintained by strict adherence to laboratory procedures designed to prevent contamination of reagents Verification of a manufacturer s lot for each type of kit used for DNA amplification will be performed using a NIST traceable control prior to use in casework analysis STANDARDS AND CONTROLS The following controls and standards are employed in the PCR procedures e There are separate locations for pre and post amplification procedures DNA extraction and PCR setup are separated by time Page 17 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 The preparation of known and questioned samples for DNA extraction is performed at different times The extraction of minimal samples is also performed at different times from other items Reagent blanks are included with each extraction to monitor the integrity of the extraction reagents and equipment This blank is carried through the amplification and typing stages Substrate controls are include
192. formation for the profile is released through the Denver laboratory DELETING CODIS PROFILES A It may be necessary to delete a record from LDAS or LDIS A hard copy of the LDAS and LDIS delete reports will be printed The analyst who performed the deletion will initial the reports and forward the record to the CODIS Administrator for that laboratory B Samples found to have been sent to the CBI in error may be removed from CODIS at the discretion of the State Administrator The State Administrator will maintain documentation supporting the removal of a sample REQUESTS FOR EXPUNGEMENT The CBI expungement procedure for the removal of DNA profiles from CODIS follows the requirements of the DNA Analysis Backlog Elimination Act of 2000 Section 6 d EXPUNGEMENT OF RECORDS of the Act provides in pertinent part 2 BY STATES A As a condition of access to the index described in subsection a referring to the national DNA index a State shall promptly expunge from that index the DNA analysis of a person included in the index by that State if the responsible agency or official of that State receives for each conviction of the person of an offense on the basis of which that analysis was or could have been included in the index a certified copy of a final court order establishing that such conviction has been overturned B For purposes of subparagraph court order is not final if time remains for an appeal or applicatio
193. formed each time the capillary array window is moved from its position Under the Tools menu select Perform Spatial Calibration If the capillaries are not yet filled with polymer this may be done under this menu by clicking the Fill Capillaries box It is not necessary to fill the capillaries each time a spatial calibration is performed Click the Start button When the first spatial calibration is finished click Cancel Per Applied Biosystems instructions the first spatial should not be accepted Perform another spatial calibration clicking off the Page 140 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 Fill Capillaries function if you have already filled the capillaries during the first spatial calibration When the second spatial calibration is finished click the details button to view the spatial If the spatial does not provide an adequate display peaks are not symmetrical or spacing between capillaries is off rerun the spatial calibration again When the spatial calibration is successful click OK to accept and save this spatial The saved spatial will be applied to all future runs until a new spatial calibration is performed For additional information regarding the procedure for a spatial calibration refer to the ABI PRISM 3100 Genetic Analyzer User s Manual 10 Perform a spectral calibration if this has not been done Spectral calib
194. from the root end of the hair Because hair may contain cellular material on the surface that may or may not originate from the hair donor it is advisable to cut off a 1 cm section of the shaft adjacent to the root portion for separate analysis as a control Alternatively the entire hair can be placed into the tube or 2 Place hair into a dish with sterile saline and while still immersed cut a 1 cm portion from the root end of the hair It is advisable to cut off a 1 cm section of the shaft adjacent to the root portion as a control To the sample add 300 yl organic stain extraction buffer 12 ul 1M DTT and 4 ul 10 mg ml Proteinase K solution Vortex on low speed for 1 second and spin in a microcentrifuge for 2 seconds to force the sample into the extraction fluid D Incubate the tube at 56 C overnight E Spin in a microcentrifuge for 2 seconds to force condensation into the bottom of the tube F In a fume hood add 300 ul phenol chloroform isoamyl alcohol to the extract Vortex low speed the mixture briefly to attain a milky emulsion Spin the tube in a microcentrifuge for 3 minutes Note If necessary additional organic extractions may be performed prior to the purification steps Page 48 of 255 CBI DNA Analysis SOP Vers 2 1 K O CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 To a Microcon concentrator add 100 ul Transfer the aqueous phase from the tube in Step F t
195. g DNA sequence This could lead to complete mischaracterization of the sample The DNA laboratories at the CBI have been organized in order to minimize the chance of any type of contamination Recommendations developed by Cetus TWGDAM DAB Roche Molecular Systems and SWGDAM have proven effective in preventing these types of contamination and have been followed here The area in which amplified DNA is handled is physically isolated from the areas for DNA extraction and PCR setup CBI has two designated work areas for DNA analysis 1 DNA extraction PCR setup room and 2 PCR amplification and typing room s The DNA extraction area is separated by time from the PCR setup area All DNA extractions are performed in the PCR workstations equipped with both fluorescent and ultraviolet lamps The PCR workstation is exposed to ultraviolet light for 20 30 minutes between DNA extraction and PCR setup between extractions of evidence samples and standard samples and between extractions of samples with minimal amounts of DNA and samples with high levels of DNA The PCR amplification and typing area is physically separated from evidence examination DNA extraction and PCR setup Production and handling of amplified DNA is restricted to the separate PCR amplification and typing room s Page 11 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 EVIDENCE EXAMINATION WORK AREA Evidence processing
196. ge 59 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 Before starting the DNA Hybridization procedure assemble the required reagents and equipment as follows QuantiBlot D17Z1 Probe provided in Kit Enzyme Conjugate HRP SA provided in Kit Hybridization Solution QuantiBlot Wash Solution Citrate Buffer 30 Hydrogen Peroxide Hybridization Tray and Lid Hybridization Tray Retainer Do NOT allow the membrane to dry at any point in the protocol Minimize the time the membrane is not submerged in solution Use the Hybridization Tray with lid for all steps Warm the Hybridization Solution and the QuantiBlot Wash Solution to between 37 and 50 C in either a water bath or an incubator AIl solids must be in solution before use Mix well NOTE Clean disposable gloves should be worn throughout the DNA Hybridi zation Section D and Detection Steps Section E 1 Pre hybridization transfer the membrane to 100 ml pre warmed Hybridization Solution in the Hybridization Tray Add 5 ml 30 H202 Place the lid on the tray Use the Hybridization Tray Retainer or a lead weight to keep tray from floating in the water bath Rotate in 50 C 1 C water bath 50 to 60 rpm for 15 minutes 2 minutes Pour off the solution 2 Hybridization add 30 ml Hybridization Solution to the Hybridization Tray containing the membrane Tilt the tray to one side and add 20 ul QuantiBlot D17Z1
197. gnostic peaks of the appropriate color and size range for a particular locus Homozygous allele peak heights are in general approximately twice that of heterozygous peaks as a result of doubling of the signal from two alleles of the same size Caution should be used with the interpretation of low RFU single allele peaks They may indicate homozygous alleles but could also be the result of preferential amplification Therefore allelic balance across the sample must be considered in the interpretation process Poor amplification at a locus may be due to degraded DNA presence of inhibitors extremely low or excessive input DNA or primer mismatch Within a single source sample genotypes generated for 0351358 075820 in the Profiler Plus and Cofiler amplification and typing systems must be concordant Page 152 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 Artificial peaks within the analysis range other than target peaks may be detected on the electropherograms These extra peaks may be caused by stutter incomplete A addition pull up spillover or spikes STUTTER The PCR amplification of tetranucleotide STR loci typically produces a minor product peak four bases shorter n 4 than the corresponding main allele peak It is also possible the case of high target DNA concentration to see n 4 addition and n 8 stutter These stutter peaks may be due to slippage during
198. guchi R Horn G T Mullis K B and Erlich H A Primer Directed Enzymatic Amplification of DNA with a Thermostable DNA Polymerase Science 239 1988 487 1 Higuchi R von Beroldingen C H Sensabaugh G F and Erlich H A DNA Typing from Single Hairs Nature 332 1988 543 6 Paabo S Gifford J A and Wilson A C Mitochondrial DNA Sequences from A 7000 year Old Brain Nucleic Acids Research 16 1988 9775 87 Hochmeister M N Budowle B Jung J Borer U V Comey C T and Dirnhofer PCR based Typing of DNA Extracted from Cigarette Butts International Journal of Legal Medicine 104 1991 229 33 Hochmeister M N Budowle B Borer U V Eggmann U Comey C T and Dirnhofer R Typing of Deoxyribonucleic Acid DNA Extracted from Compact Bone from Human Remains Journal of Forensic Sciences 36 1991 1649 61 Sajantila A Strom M Budowle B Karhunen P J and Peltonen L The Polymerase Chain Reaction and Post mortem Forensic Identity Testing Application of Amplified D1S80 and HLA DQ Alpha Loci to the Identification of Fire Victims Forensic Science International 51 1991 23 34 Blake E Mihalovich J Higuchi R Walsh P S and Erlich H Polymerase Chain Reaction PCR Amplification and Human Leukocyte Antigen HLA DQa Oligonucleotide Typing on Biological Evidence Samples Casework Experience Journal of Forensic Sciences 37 1992 700 26 Comey
199. h care 11 28 The dots on the AmpliType HLA DQA1 DNA Probe Strip correspond to the following alleles The 1 dot is positive in the presence of the HLA DQA1 1 1 1 2 and 1 3 alleles The 2 dot is positive only in the presence of the HLA DQA1 2 allele The 3 dot is positive only in the presence of the HLA DQA1 allele The 4 dot is positive in the presence of the HLA DQA1 4 1 4 2 and 4 3 alleles Four HLA DQA1 sub typing probes differentiate the HLA DQA1 1 1 1 2 and 1 3 alleles Page 80 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 The 1 1 dot is positive only in the presence of the HLA DQA1 1 1 allele NOTE A faint 1 1 dot will appear with some HLA DQA2 pseudogene alleles The 1 3 dot is positive only the presence of HLA DQA1 1 3 allele NOTE There is no probe that detects only the HLA DQA1 1 2 allele The 1 2 1 3 4 dot is positive in the presence of 1 2 1 3 4 1 4 2 and 4 3 alleles NOTE The 1 2 1 3 4 dot can be lighter than the C dot when the genotype has HLA DQA1 4 2 or 4 3 allele paired with HLA DQAf1 1 1 2 3 4 2 or 4 3 allele because the HLA DQA1 4 2 and 4 3 alleles each have a single partially destabilizing mismatch to the 1 2 1 3 4 partially destabilizing mismatch allows these two alleles to bind to this probe weakly relative t
200. h locus are similar Reading and Interpreting the AmpliType HLA DQA1 DNA Probe Strips The AmpliType HLA DQA1 DNA Probe Strips have been spotted with a total of eleven sequence specific oligonucleotide probes to detect eight alleles of the HLA DQA1 locus Under the AmpliType hybridization conditions the typing probes will bind specifically to PCR product containing the alleles designated on the AmpliType HLA DQA1 DNA Probe Strip To read the developed AmpliType HLA DQA1 DNA Probe Strip the C dot is examined first and then the remaining dots are examined The control probe C on the AmpliType HLA DQA1 DNA Probe Strip detects all of the HLA DQA1 alleles The C dot is designed to be the lightest typing dot on the strip and it indicates that adequate amplification and typing of the HLA DQA1 alleles in the sample have occurred If the C dot is absent an accurate determination of the type cannot be made Additional information on the C dot can be found in the AmpliType User Guide 1 The accurate interpretation of the HLA DQA1 results depends on the presence and intensity of the C dot The intensities of the dots at the remaining ten positions are compared to the intensity of the C dot Those dots that appear either darker than or equivalent to the C dot are considered positive Each positive dot indicates the presence of the corresponding HLA DQA 1 allele Dots with signals less than the C dot should be interpreted wit
201. h sample approximately 155 ul into a different well of the slot blot apparatus Slowly dispense each sample directly into the center of each well of the slot blot apparatus ensuring that the pipette tip is approximately 5mm above the membrane 8 After all samples have been pipetted into the wells of the slot blot apparatus slowly turn on the sample vacuum Leave the sample vacuum on until all of the samples have been drawn through the membrane approximately 30 seconds Inspect each slot that contains a sample for a uniform blue band If a uniform blue band is not visible refer to Section T Turn off the sample vacuum 9 Turn off the clamp vacuum Turn off the vacuum source Disassemble the slot blot apparatus and remove the membrane Proceed to Section D immediately Do NOT allow the membrane to dry out NOTE After each use soak the slot blot apparatus in a large volume of 0 1 SDS solution approximately 5 to 15 minutes Using a disposable lab towel clean the gasket and the side of the top plate that contacts the membrane Then rinse the slot blot apparatus with an excess of water and allow to dry at room temperature Never use bleach D DNA Hybridization The following section involves the hybridization of biotinylated QuantiBlot 01721 Probe to DNA samples immobilized on the nylon membrane the binding of Enzyme Conjugate HRP SA to the hybridized probe and a stringent wash to remove non specifically bound probe Pa
202. he analyst s discretion REAGENT BLANKS A reagent blank tests for possible presence of contamination of the extraction reagents and or supplies by an adventitious source of DNA The adventitious source of DNA may be non amplified DNA or PCR product Peaks gt 50 RFU not attributable to artifacts located between 100 and 350 bp or at 75 bp and 400 bp indicate the presence of contamination and none of the samples extracted or amplified with the reagent blank will be considered for analysis interpretation The samples should be reamplified and reanalyzed NEGATIVE AMPLIFICATION BLANKS A negative amplification blank is a test for the possible presence of contamination occurring during the amplification setup If a negative amplification blank exhibits peaks 250 RFU not attributable to artifacts located between 100 and 350 bp or at 75 bp and 400 bp this indicates the presence of contamination and none of the samples extracted or amplified with the negative amplification blank will be considered inclusive for analysis interpretation The samples should be reamplified and reanalyzed POSITIVE CONTROLS The positive control should have all the alleles present using a 50 RFU cut off They must be genotyped correctly If alleles are missing or mistyped the analysis run must be repeated If this does not solve the problem the entire set of samples should be reamplified and retyped MERGING GENOTYPER ALLELIC TABLE FILES AFTER TECHNICAL REVIEW Th
203. he block valves and ferrule thoroughly with warm tap water if necessary then with filter purified water 6 Remove any excess water from inside and outside of the block using compressed air 7 Verify that the gold electrode socket on the back of the block is dry and then replace the pump block Slide the U shaped end of the activator arm into the collar at the top of the plunger valve Make sure the activator arm lines up with the groove in the pin valve before pressing into place It may be necessary to move the activator arm up or down by selecting Buffer Valve Open or Closed from the Function pop up menu and selecting Execute K Installing the Syringe on the Pump Block 1 Move the syringe drive toggle on the subject pump drive to the left to allow for replacement of the syringe to the pump block 2 Place the 1 0 syringe in through the right hand port of the syringe guide plate and screw it into the pump block The syringe should be finger tight in the block NOTE Tighten the syringe at the metal collar Excess pressure on the glass syringe barrel will break the seal of the collar permanently damaging the syringe 3 Hand tighten the valves on the pump block to the left of and below the syringe L Installation of a New Capillary 1 Remove the new capillary from the plastic sleeve and clean the detection window with ethanol and lens paper 2 Open the door covering the heat plate and then partially unscrew the plastic
204. he sample vacu um slowly not all at once After being drawn through the membrane the sam ple should appear as a uniform blue band on the membrane If the entire sample is not drawn through the mem brane turn off the sample vacuum Pipet the sample back into the pipette tip then pipet the sample back into the well of the slot blot apparatus Turn on the sample vacuum to draw the sample through the membrane Prepare solutions with proper concentrations of SDS Pre wet the membrane in Pre Wetting Solution prior to slot blotting Use 180 ul Enzyme Conjugate HRP SA for colorimetric detection CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 OBSERVATION POSSIBLE CAUSE RECOMMENDED ACTION Lack of thorough rinsing at Thoroughly rinse twice Section 0 5 of the DNA for 1 minute each using Hybridization protocol 100 pre warmed Wash Solution at this step These two rinse times can be extended beyond 1 minute if nec essary Slot blot apparatus not Immediately after each cleaned thoroughly after last use soak the slot blot use apparatus in a large vol ume of 0 1 SDS solution Never use bleach 5 The DNA Calibrators do DNA Standard serial dilu Prepare two fold serial not quantitate correctly tions prepared incorrectly dilutions of DNA Standard with respect to the DNA A in TE Buffer as de Standards scribed Add 5 yl of each dilution to 150 ul Spotting Solution for slot blotting
205. hould be greater than 9 Date Amount Chelex lot Preparer Verified by initials date Prepared CHELEX20 Page 232 of 255 Appendix Y DNA Working Solutions Log 20 w v Chelex Solution CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 APPENDIX Z DNA WORKING SOLUTIONS Loc DEIONIZED FORMAMIDE MODEL 50 Solution Preparation One of the following two methods can be employed 1 Aliquot Hi Di Formamide ABI Cat 4311320 into microcentrifuge tubes Store at 20 C Use only if lot freezes 2 Mix 50 Formamide and 5 Dowex MR3 Sigma Cat 19005 ion exchange resin Stir for 30 minutes at room temperature Check that the pH is greater than 7 If the pH is less than 7 start the process again utilizing the same formamide but different Dowex MR3 ion exchange resin Allow the resin to settle Filter the formamide and aliquot into 1 5 ml vials Store up to 3 months at 20 C Date Preparation Amount Formamide lot Dowex MR3 resin lot Preparer FORMAMIDE Page 233 of 255 Appendix Z DNA Working Solutions Log Deionized Formamide CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 APPENDIX AA DNA WORKING SOLUTIONS Loc DIGEST BUFFER MODEL 10MM TRIS HCL 10MM EDTA 50MM NACL 2 SDS 7 5 100
206. iluminescent Method for Quantification of Human DNA Nuc Acids Res 20 1992 5061 5 Waye J et al A Simple and Sensitive Method for Quantifying Human Genomic DNA in Forensic Specimen Extracts Bio Techniques 7 8 1989 852 5 POLYMERASE CHAIN REACTION Cimino G D Metchette K C Tessman J W Hearst J E Issacs S T Post PCR Sterilization A Method to Control Carryover Contamination for the Polymerase Chain Reaction Nuc Acids Res 19 1991 99 107 Dieffenbach C W Dveksler G S Setting Up a PCR Laboratory PCR Meth amp Appl Suppl 3 1993 52 57 Dragon E Handling Reagents in the PCR Laboratory PCR Meth amp Appl Suppl 3 1993 58 59 Page 190 of 255 Appendix C Resources CBI DNA Analysis SOP Vers 2 1 06 13 02 4 43 PM CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 Ellingboe James and Gyllensten Ulf B eds The PCR Technique DNA Sequencing Natick MA Eaton Publishing Co 1992 Erlich Henry A ed PCR Technology Principles and Applications for DNA Amplification New York Stockton Press 1989 Erlich H A Gelfand D Sninsky J J Recent Advances in the Polymerase Chain Reaction Science 252 1991 1643 51 Haff L Atwood J G DiCasare J Katz E Picozza E Williams J F Woudenberg T A High performance System for Automation of the Polymerase Chain Reaction Biotechniques 10 1991 102 12 Hartley J Rashtchian A
207. including examination and sample collection is performed in the Serology laboratory Stain cards from database samples are prepared in a hood DNA EXTRACTION AREA All DNA extractions are performed in the PCR workstations located in the DNA extraction PCR setup room of the laboratory The following special precautions are strictly observed e Evidence samples are prepared at a separate time from standard samples Samples with minimal amounts of DNA are prepared at a separate time from samples with high levels of DNA e Disposable gloves are worn during evidence examinations and analyses Gloves are changed as necessary to avoid sample to sample contamination and when leaving the work areas Sampling instruments are cleaned thoroughly with 10 bleach and deionized water between uses A clean cutting surface is used sheet of paper for each specimen and absorbent hood pads are replaced as necessary Sampling is limited to that amount from which might expect to obtain sufficient DNA as required by the investigation at hand Those solutions which can be heated in an autoclave without affecting their performance are steam sterilized This insures that any contaminating DNA is rendered unamplifiable e Sterilized microcentrifuge tubes and sterile aerosol resistant pipette tips are used e Aerosol resistant pipette tips are changed between each sample They do not need to be changed when aliquotting kit reagents
208. ing Norton Utilities to insure efficient computer operation QUALITY CONTROL OF CRITICAL SUPPLIES AND REAGENTS Prior to being used for case analysis certain supplies and reagents should be checked against materials known to give an acceptable result The testing procedures for these items are given below AMPLIFICATION REAGENTS Profiler Plus and Cofiler Kits Each lot of kits will be evaluated using the extraction control that has been designated to be NIST traceable The criteria for kit quality sufficient for use in casework are A a minimum of 0 75 ng sensitivity and B correct genotypes obtained for the NIST traceable CBI internal control or extraction control The genotypes for the NIST traceable CBI DNA controls will be kept in the logbook with the results of the kit validations The positive amplification control 9947A will be suitable for use in case work if complete and correct typing of this sample is obtained The allelic ladders are suitable for use in case work if C each ladder allele is greater than 100 RFU and D correct typing results are obtained for 9947A when the ladders are employed in Genotyper QUANTIFICATION PROCEDURE An estimation of the DNA content of the samples will be conducted prior to STR analysis CBI uses the QuantiBlot biotin streptavidin HRP method to evaluate the DNA concentration of each extracted sample In general 0 75 to 2 0 ng of DNA is optimal for STR amplification although correct
209. internal lane standard ROX 500XL with DNA fragments of known size Sample peaks are assigned a size by interpolation The ABI 3100 Genetic Analyzer uses a Windows NT computer platform to collect raw data and also uses Genescan analysis software A matrix created from the spectral calibration of the 3100 is applied at the point of data collection to correct for spectral overlap A size curve is created using an internal size standard GS 500 LIZ GS 500ROX with DNA fragments of known size sample peaks are assigned a size by interpolation A new matrix will be created for each instrument as needed i e if the matrix in use consistently fails to eliminate pull up or over subtracts one or more colors Refer to Multicomponent Analysis in the Cofiler Profiler Plus or Identifiler User Manuals for instructions on how to build a matrix Once prepared a matrix will be considered suitable for database analysis if a flat baseline is obtained when it is applied to the matrix standards With the ABI 3100 Genetic Analyzer certain capillaries can fail spectral calibrations and thus fail to produce matrices These capillaries can borrow the spectral calibrations of adjacent capillaries that have passed their spectral calibrations At least twelve capillaries should produce matrices If not repeat the spectral calibration process until at least twelve capillaries pass NOTE A new matrix must be built following cleaning maintenance or replaceme
210. les DESKTOP COMPUTERS Desktop computers in the amplification room are used to run the instrumentation and collect data Desktop computers in office areas contain software used to analyze data and obtain DNA profiles for entry into the CODIS database QUALITY CONTROL OF CRITICAL REAGENTS For database analysis critical reagents should be checked against materials known to give an acceptable result The quality control requirements for these items are given below PROFILER PLUS COFILER AND IDENTIFILER KITS Each lot of kits will be verified using a NIST traceable internal DNA control The criterion for kit quality sufficient for use in database analysis is that a correct genotype must be obtained for the internal DNA control used Kit lots need not be verified prior to database analysis Page 129 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 FTA PAPER New lots of FTA paper should be verified prior to use on any database sample To verify the new lot a sample is spotted on stain cards from both the new and old lots Identical profiles must be obtained through PCR profiling Peak heights and peak resolution should be similar If this does not occur a different lot of FTA paper should be obtained EXTRACTION PROCEDURE Database samples are extracted with FTA and Chelex procedures See Inorganic and FTA extraction procedures in this SOP for details QUANTIFI
211. liType PM DNA Probe Strip 8 Some but not all loci visible on strips JUNE 2002 POSSIBLE CAUSE Amplification of DQA2 pseudogene faint 1 1 dot EDTA not added to the reaction prior to the heat denaturation step of the DNA hybridization proto col Cross hybridization caused by Hybridization and or Wash temperature too low Cross hybridization caused by Hybridization and or Stringent PM DQA1 Wash Solution salt concentration too high Stringent Wash time too short Mixed sample or contami nation Amplification of DQA2 pseudogene faint 1 1 dot Degraded input DNA Page 86 of 255 CBI DNA Analysis SOP Vers 2 1 RECOMMENDED ACTION See References 18 and 30 Add EDTA to amplified sample Section PREPA RATION OF PCR DUCT FOR DETECTION repeat test Check that rotating water bath temperature is at 55 C 1 C repeat test Prepare solutions repeat test new Repeat test washing for 12 minutes 1 minute See References 14 15 17 18 and 28 See References 18 and 30 Evaluate the quality of the DNA sample If the DNA is degraded re amplify with an increased amount of DNA CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 OBSERVATION 9 Imbalanced dot in tensities within a locus the kit is designed to produce balanced dot intensities when het erozygous samples are typed except as described in Observa ti
212. lic ladders provided in the kit are used to determine the genotypes of the sample Page 127 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 GUIDELINES FOR CONTROL SAMPLES It is imperative that proper control samples be included when database samples are extracted quantified where appropriate amplified and typed The typing results obtained from these control samples are essential for the interpretation of STR and amelogenin typing results from database samples The controls also serve to assess the quantity and quality of the extracted DNA as well as the effectiveness accuracy and precision of the analytical procedures EXTRACTION CONTROLS Reagent Blank With each database extraction set of samples a reagent blank RB must be included The reagent blank consists of all reagents used in the test process minus any sample The reagent blank is processed through the entire extraction quantification where appropriate amplification and electrophoretic typing procedure With FTA extractions the reagent blank tube is subjected to the same extraction conditions as the samples The reagent blank must be run in all PCR systems to check for contamination of the reagents with extraneous DNA AMPLIFICATION CONTROLS Negative Amplification Blank A negative amplification blank NAB must be used with each amplification The negative amplification blank contains all components required
213. listing the file names specimen names and whether each specimen is accepted or rejected Any specimen that is rejected must be examined to resolve any discrepancies The explanation for the rejection and any further actions should be noted on the report and initialed by both analysts CREATING CMF FILES A CMF file is a common message format file which contains DNA data in a format suitable for electronic transfer into CODIS The data includes the specimen name analyst s name laboratory ORI index name and allele calls for each database profile The following are the two procedures in use at the CBI for creating CMF files CREATING A CMF FILE USING NT SOFTWARE The Windows NT CMF macro is used to merge the analyst s and reviewer s tables create a report listing each sample and whether it was accepted or rejected and convert the information for import into CODIS Thus it serves two purposes it is used as a part of the technical review and then the same file is used to electronically import data into CODIS A Select the CMF convert icon on the desktop or within Excel The following information is displayed File Info Source Lab enter your lab ORI Destination Lab enter your lab ORI Read by Analysts CODIS user name Input Input file 1 Analysts CODIS table Input file 2 Reviewer s CODIS table Output Save as dateanaCMF Error Handling Select prompt for action for both statements Page 157 of 255 CBI DNA Analysis SOP
214. ls Seat the syringe drive 1 2 Select Buffer Valve Open from the Function pop up menu and click Execute Move the syringe toggle to the right so that it is positioned over the syringe plunger Select Syringe Down from the Function pop up menu and enter a value from 200 to 5 and click Execute Repeat this procedure until the toggle seats on the plunger Use smaller increments the closer the toggle comes to the plunger Page 105 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 Set the temperature 1 Close the instrument doors 2 Select Temperature Set from the Function pop up menu and enter 60 in the Value box Click Execute It takes approximately 30 minutes for the instrument to reach the run temperature of 60 C PREPARING FOR A RUN WARNING CHEMICAL HAZARD Formamide is an irritant and a teratogen Avoid skin contact and inhalation Use in a well ventilated area Wear lab coat and gloves when handling A B Remove deionized formamide from the freezer to thaw Prepare a sample sheet 1 Choose New from the file menu 2 Click on the appropriate GeneScan Sample Sheet icon Fill in the appropriate information as defined by current software directions 3 Fill in the sample name column first When complete highlight the Sample Name column by clicking once in the gray bar at the top and select Copy from the Edit menu Highlight the Sample Info co
215. ltrapure water and mix thoroughly Note Hybridization solution solids must be in solution before use warming e g in 50 C incubator may be required to dissolve solids completely Preparation in a clear glass container is recommended to facilitate visual inspection for solids during warming Date Prepared Amount probo m 20 SDS Preparer HYBSOLN Page 246 of 255 Appendix AM DNA Working Solutions Log QuantiBlot PM DQA1 Hybridization Solution CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 APPENDIX AN DNA WORKING SOLUTIONS LOG 20 SARKOSYL MODEL N LAURYLSARCOSINE 100 ML Solution Preparation Add 20 N laurylsarcosine C15H2gNO3Na to ultrapure water and stir until dissolved Bring to a final volume of 100 ml with ultrapure water and sterilize by passage through a 0 45 u sterile filter Date Prepared AMOUNT N laurylsarcosine lot Preparer Verified by initials date 20 SARKOSYL Page 247 of 255 Appendix AN DNA Working Solutions Log 2096 Sarkosyl CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DN
216. lumn and select Paste from the Edit menu Insure there is a diamond next to the red box in the Std column for each sample 6 Make sure there is an X in each column for the Pres column NOTE The sample name must be in accordance with the guidelines in CODIS Standard Operating Procedures Refer to CODIS Identification Number in Section Insure that the information contained under the Sample Name column is copied onto the Sample Info column See section Electronic File Naming Convention for the 310 STR Analysis Note Insure that the Std column highlights red Note Under the Sample Info column insure that each sample has a unique sample number also insure that each lane which contains an allelic ladder has the word ladder in the Sample Info column this is necessary for automated allele calling with Genotyper v 2 0 or higher 7 Select Save As from the File menu and label the sample sheet SS date analysts initials PP and or eg SS01 22 00xyzPP CO SS012200xyzPPCO Page 106 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 Prepare the samples for analysis 1 11 Remove the GeneScan 500 ROX internal lane standard and allelic ladders from the refrigerator Vortex and spin Add GeneScan 500 ROX to the thawed formamide at a concentration necessary to obtain an RFU of 750 to 3000 in the blank Vortex the tube and spin in a cent
217. ly between 20 ul and 200 ul J Spin the assembly in a microcentrifuge at 2500 x g for 5 minutes K Discard the concentrator Cap the retentate cup L Estimate the quantity of DNA in the sample by slot blot hybridization Page 45 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 M After quantification the sample can be amplified N Store samples at 4 C or frozen Prior to the use of samples after storage they should be vortexed briefly and spun in a microcentrifuge for 5 seconds CIGARETTE BUTTS A Collect a portion of the filter and or paper of the cigarette butt in the area that would have been in contact with the mouth B Cut into smaller pieces and place into a 2 2 ml microcentrifuge tube To the sample add 300 yl organic stain extraction buffer 12 ul 1M DTT and 4 ul 10 mg ml Proteinase K solution Vortex for 1 second and spin in a microcentrifuge for 2 seconds to force the butt into the extraction fluid D Incubate the tube at 56 C overnight E Spin in a microcentrifuge for 2 seconds to force condensation into the bottom of the tube F In a fume hood add 300 ul phenol chloroform isoamyl alcohol to the stain extract Vortex the mixture briefly to attain a milky emulsion Spin the tube in a microcentrifuge for 3 minutes Note If necessary additional organic extractions may be performed prior to the purification steps G Assemble a Microcon con
218. ly label many of the alleles not present in the allelic ladders however manual genotyping may be used to determine a genotype where Genotyper does not make the assignment Manual genotypes are assigned using the base pair calculations by the GeneScan software Whenever an off ladder allele is considered a true off ladder allele a variant allele rather than an off ladder allele due to spikes pull up split peaks etc it must be confirmed by re amplification and re typing if it conforms to the same overall guidelines described in the Interpretation of Data During the course of analysis if the same off ladder allele occurs in two or more different sample amplifications then re amplification Page 114 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 and re typing are not necessary The existence of a microvariant is confirmed by its presence in the multiple samples Off ladder alleles OLA that fall between alleles within the ladder will be designated in accordance with guidelines of the International Society of Forensic Haemogenetics OLA calls are first converted to sizes in base pairs then compared to the size of the appropriate ladder alleles and the allelic designation determined If the OLA is not a perfect repeat but rather varies by 1 2 or 3 base pairs from a ladder allele then it will be designated as an integer of that variation For example if a green OLA peak size is 238 8
219. ly resembles samples extracted It is extracted along with the casework samples using the same reagents and is NIST Page 96 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 traceable The alleles for each locus are documented in the CBI Forensic Laboratory NIST Logbook AMPLIFICATION CONTROLS Negative Amplification Blank A Negative Amplification Blank must be used with each set of amplifications The negative amplification blank contains all components required for the amplification of DNA except that no DNA is added Twenty microliters of TE Buffer is placed in the negative amplification blank in lieu of a DNA solution This control is processed through the amplification and capillary electrophoretic typing procedures Positive Amplification Controls Cell line DNA 9947A is a positive control for STRs and amelogenin to evaluate the performance of amplification and capillary electrophoresis When the control specimen 9947A is amplified the STR loci must solely exhibit the correct genotype Additionally specimen 9947A is the female control for amelogenin and must exhibit a single band at the position corresponding with the size ladder band representative of the 103 bp peak from the X chromosome EQUIPMENT Thermal Cycler 480 and 9700 The DNA thermal cycler 480 or 9700 can be used to amplify both Chelex extracted and organic extracted casework samples the thermal cycler is ev
220. m is examined on clean paper generation of blood dust is minimized and tools and work area are properly cleaned after each use This eliminates the potential for one item to contaminate another through casual contact especially in those instances where large differences in potentially recoverable DNA exist between items B Sampling of evidence for conventional serological analysis must be performed with consideration of whether DNA analysis could be more informative or appropriate C Prior to DNA processing hair should be microscopically characterized MISCELLANEOUS EVIDENCE In addition to blood semen and saliva evidence PCR technology can be utilized for the analysis of many other potential evidence specimens such as cigarette butts postage stamps and envelopes dry bone preserved autopsy specimens etc MINIMAL EVIDENCE SUBMITTED FOR DNA ANALYSIS Evidence submitted to the DNA section may be minimal in nature and completely consumed during analysis In the event that upon evaluating a case the analyst feels that any item might be consumed in the analysis the analyst shall not proceed with the analysis of those items The District Attorney and the submitting agency in that specific case shall be notified in the report of the nature of the minimal evidence The District Attorney and the submitting agency may also be notified by telephone of the issue Analysis of minimal evidence shall only proceed upon the written authorization of
221. mM 2 96 SDS pH 8 0 500 ml Add 5 ml 1M Tris HCl pH 8 0 10 0 5M EDTA pH 8 0 5 5M NaCl and 50 20 SDS to 430 ml ultrapure water or use 0 29 NaCl in place of the 5 ml 5M NaCl and adjust volume to 500 ml The pH should be adjusted to 8 0 Autoclave Store at room temperature Page 28 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 ORGANIC STAIN EXTRACTION BUFFER 10mM Tris 100mM NaCl 10 EDTA 2 SDS 1L Dissolve 5 84 g NaCl 500 ml ultrapure water with stirring To this solution add 10 ml 1M Tris pH 8 0 20 ml 0 5M EDTA and 100 ml 20 SDS Titrate to pH 8 0 with Bring to a final volume of 1L with ultrapure water Autoclave Store at room temperature PBS BUFFER pH 7 4 PHOSPHATE BUFFERED SALINE 2 7mM KCI 137mM NaCl 1 5mM 4 8 0mM NazHPO pH 7 4 1L Dissolve 0 2 g KCI 8 0 NaCl 0 2 KH PO and 2 2 Na HPO e7H2O or 1 1 g 4 anhydrous 800 ml ultrapure water Adjust pH of solution to 7 4 if necessary Adjust to final volume of 1 liter using ultrapure water Sterilize by autoclaving Store at room temperature Phenol chloroform isoamyl alcohol 25 24 1 v v Phenol chloroform isoamyl alcohol saturated with buffer to a pH of 8 0 0 5 is used for the purification of DNA CAUTION This solution is an irritant and is toxic it must be confined to a fume hood
222. maining blood and blood tube are destroyed after a stain card is prepared Stain cards are stored at CBI according to the preparation procedure utilized No database samples are released from the CBI Laboratory PREPARATION OF SAMPLES FOR DNA ANALYSIS PRESERVATION Evidence materials must be stored handled and processed keeping in mind the potential for conventional serological and DNA analysis Evidence will be stored according to existing preservation procedures in the Laboratory to insure that DNA genetic evidence as well as conventional serology evidence will be preserved while in custody at CBI General identification of the biological material is performed prior to DNA analysis Evidence samples submitted are evaluated to determine the appropriateness for DNA analysis Testing of evidence and evidence samples should be conducted to provide the maximum information with the least consumption of sample It is necessary to preserve a portion of the sample for future analysis unless written permission to consume the entire sample is obtained The procedures in place at the CBI in regard to the conventional examination sampling and analysis of biological evidence are applicable also for DNA analysis of such evidence Some of the guidelines with special pertinence to DNA analysis of evidence are reiterated below Page 15 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 A Each ite
223. n for 3 minutes Click on the Pause button to pause the Pre Run Rinse the wells with 1X TBE Load all even wells in sequence Run the gel T Terminate the Pre Run 2 Click on the Run module and enter a file name Start the run At the end of the run save the Run Folder with the date analyst s initials and PP or CO e g Run Folder mm dd yyanaPP CO Transfer the Run Folder to an analysis Macintosh for review Clean the 377 1 2 Open the door to the instrument Remove the heat plate Remove the lid to the upper buffer chamber Rinse the lid with ultrapure water Suction out the buffer from the upper and lower buffer chambers Use separate devices to avoid contaminating the upper buffer chamber with excess fluorescent dyes Remove the upper buffer chamber and rinse it with ultrapure water Remove the gel cassette Unclamp the glass plates Rinse the gel cassette with hot tap water followed by ultrapure water Disassemble the glass plates Lay a Kimwipe across the plate to remove the gel Soak the glass plates in hot tap water to remove any remaining acrylamide Rinse them thoroughly with hot tap water followed by ultrapure water and allow them to air dry in a plate rack Rinse the spacers in hot water followed by ultrapure water USE ONLY WATER TO CLEAN THE GLASS PLATES DETERGENT OR ETHANOL MAY CONTAIN FLUORESCENT PARTICLES THAT COULD ADHERE TO THE GLASS AND CAUSE BACKGROUND FLUORESCENCE Page 138 of 255 CBI DNA Anal
224. n for discretionary review with respect to the order Page 173 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 Therefore CBI requires both a written request and a certified copy of a final court order as defined above to implement the expungement procedure The written request and the court order directing the expungement must be issued to CBI It then must be reviewed and verified by the AIC of the DNA section to which the order was directed prior to proceeding with expungement Upon this approval the CODIS Administrator of the laboratory that entered the record shall be responsible for A E F Deleting all DNA profiles records and identifiable information in the CODIS database pertaining to the person with regard to the dismissed or reversed conviction Destroying all samples obtained from the individual that pertain to the dismissed or reversed conviction Deleting the profile and sample name from the appropriate DNA analysis files For convicted offenders deleting the name date of birth sex and DOC ML DYC identifiers from the Offender Inventory Log Replace the name with NO RECORD EXPUNGED Place the date of expungement under date of birth DO NOT RELEASE ANY INFORMATION FOR THE SAMPLE A NO RECORD RESPONSE MUST BE GIVEN Placing a copy of the expungement order in the case file Notifying the State CODIS Administrator and provide a copy for the state r
225. nalysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 This Page Intentionally Left Blank Page 176 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 DNA DATABASE Database DBS samples may consist of whole blood buccal swabs or prepared cards required for genetic testing mandated by legislation DBS submissions are received from the Department of Corrections Probation Division of Youth Corrections and Community Corrections for entry into the DNA database only No report is generated and submitted samples are not returned SUBMISSION PREPARATION AND STORAGE OF SAMPLES FOR OFFENDER DATABASE A Submission Procedures 1 Samples a Blood samples must consist of at least one 4ml Lavender purple top EDTA tube from each offender Buccal samples must be dried on the appropriate substrate and then sealed in a protective package 2 Submission Labeling And Packaging a The blood tube is labeled with the offender s name first amp last The submitting agency case number date the blood was drawn and the initials of the person collecting the sample are additional identifiers that are preferred but not required If no identifiers are present on the blood tube the tube is not analyzed and a redraw request is made The protective packaging containing the buccal sample is labeled with the offenders name first amp last The submitti
226. nation of Peroxidase Labeled Conjugates in Immunoassay Nature 305 1983 158 9 Miller S A Dykes D D and Polesky H F A Simple Salting Out Procedure for Extracting DNA from Human Nucleated Cells Nucleic Acids Research 16 1988 1215 Page 66 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 This Page Intentionally Left Blank Page 67 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 AMPLITYPE DQA1 STORAGE AND STABILITY A Store the AmpliType PM DQA1 PCR Amplification and Typing Kit components at 2 to 8 C Isolate the kit from any sources of contaminating DNA especially amplified PCR product B Store the AmpliType DNA Probe Strips protected from light at 2 to 8 C Store the DNA Probe Strips with the desiccant in the glass tube and insure that the screw cap is securely tightened C Under these conditions components of the kit are stable through the control date printed on the label D The Chromogen Solution is stable for 6 months after its preparation when stored at 2 to 8 C PREPARATION OF REAGENTS SUPPLIED A Chromogen TMB Solution Bring the bottle of Chromogen TMB TMB to room temperature 15 to 30 C Before opening the bottle tap it on the lab bench to shake the TMB to the bottom of the bottle Remove the stopper carefully to prevent loss of the powder Slowly add
227. nd and spin in a microcentrifuge for 2 seconds to force the cutting into the extraction fluid C Incubate the tube at 56 C overnight D Spin in a microcentrifuge for 2 seconds to force condensation into the bottom of the tube In fume hood add 300 ul phenol chloroform isoamyl alcohol to the stain extract Vortex the mixture briefly to attain a milky emulsion Spin the tube in a microcentrifuge for 3 minutes Note If necessary additional organic extractions may be performed prior to the purification steps Assemble Microcon concentrator unit the top of the concentrator 100 ul Transfer the aqueous phase from the tube in Step E to the top of the concentrator Avoid pipetting organic solvent from the tube into the concentrator G Place a cap on the concentrator and spin in a microcentrifuge at 2500 x g for 10 minutes H Add 100 TE to the concentrator Replace the cap and spin the assembly in a microcentrifuge at 2500 x g for 10 minutes l Remove the cap and add a measured volume of that is between 20 ul and 200 ul to the concentrator Remove the concentrator from the filtrate cup and carefully invert the concentrator onto a labeled retentate cup Discard the filtrate cup Note Final recovery volumes following purification generally range from 20 ul to 200 ul for evidentiary samples The final recovery volumes for the extraction reagent blanks for evidentiary samples are general
228. ng Trays H Perform the following steps for each tube of amplified DNA NOTE For each tube perform Steps H 1 through H 3 within 20 seconds 1 Remove the tube from the 95 C block of the GeneAmp PCR Instrument System 2 Carefully open the tube using the microcentrifuge tube de capping device designated for use only with amplified DNA 3 Withdraw 20 ul of amplified DNA from the aqueous bottom layer and immediately add it below the surface of the hybridization solution in the well of the corresponding probe strip see Figure 1 4 Cap the tube after adding the denatured amplified DNA 5 Repeat Steps H 1 through H 4 until each amplified DNA sample has been added to the corresponding well The remaining amplified DNA samples can be stored at 2 to 8 C for two weeks or at 20 C for 6 months The continued acceptable performance of these samples beyond these periods may vary with the sample Store amplified DNA samples separate from all PCR amplification reagents extracted DNA samples and casework samples l Put the clear plastic lid on the tray and mix by carefully rocking the tray Insure that each strip is completely wet Once hybridization has begun strips should remain wet through the conclusion of the Color Development and Photography steps Page 74 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 J Transfer the tray to the 55 C water bath Place a 1
229. ng agency case number the date the sample was collected and the initials of the person collecting the sample are additional identifiers that preferred but not required If no identifiers are present on the saliva sample the sample is not analyzed and a request for another sample is made The CBI Database Submission Form must accompany DBS sample The form will be kept separate from the blood tube or buccal sample and its sealed container If a blood tube is broken or leaking at time of submission to CBI the sample will not be accepted or will be destroyed If the sample does not meet the above requirements personnel will use discretion as to whether the sample will be analyzed At the time of sample submission to the Denver Laboratory signature is required CBI Personnel an evidence technicians an Page 177 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 AIC or a criminal investigator assigned to the database section will stamp the chain of custody with the date received and assign a case number to each sample Once the samples have been assigned a case number they are labeled and stored g Samples requiring refrigeration will be stored in a refrigerator located in the CBI Serology section CBI HANDLING OF DATABASE SAMPLE SUBMISSIONS A Data Entry 1 The CBI Database Submission Forms are checked for errors and omissions Name date
230. nsic Short Tandem Repeat STR Systems Based on Sizing Precision in a Capillary Electrophoresis Instrument Electrophoresis 19 1998 86 93 Li H Schmidt L Wei M H Hustad T Lerman Abar B and Tory K Three Tetranucleotide Polymorphisms for Loci D3S1252 D3S1358 D3S1359 Hum Mol Genet 3 1993 1327 Mayrand P E Corcoran K P Ziegle J S Robertson J M Hoff L B and Kronick M N The Use of Fluorescence Detection and Internal Lane Standards to Size PCR Products Automatically Applied and Theoretical Electrophoresis 3 1992 1 11 Mills K A Even D and Murray J C Tetranucleotide Repeat Polymorphism at the Human Alpha Fibrinogen Locus Hum Mol Genet 1 1992 779 Moller A Meyer E and Brinkmann B Different Types of Structural Variation in STRs Hum FES FPS Hum vWA and HumD2S11 Intl J Leg Med 106 1994 319 23 Polymeropoulos M H Rath D S Xiao H and Merrill C R Tetranucleotide Repeat Polymorphism at the Human Tyrosine Hydroxylase Gene TH Nucleic Acid Res 19 1991c 3753 Puers C Hammon H Jin L Caskey C and Schumm J Identification of Repeat Sequence Heterogeneity at the Polymorphic Short Tandem Repeat Locus HUMTH01 AATG and Reassignment of Alleles in Population Analysis by Using a Locus specific Allelic Ladder Am J Hum Genet 1 1 1992 67 Sharma V and Litt Tetranucleotide Re
231. nt of a component of the optics or CCD camera GENESCAN ANALYSIS USING MACINTOSH SOFTWARE A Open the Run Folder and locate the Gel File Open the Gel File and check the tracking to insure accuracy Retrack if necessary then re extract the data and close the file B Open the GeneScan Project Page 145 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 C Within the Analysis Control Window choose the blue green yellow and red colors for analysis Also insure that the red dye color is marked as the size standard a diamond symbol should appear in the red boxes D Insure that the analysis parameters are as below 1 Analysis Range Select a range of data points incorporating both the 75 and 400 bp size standards Data Processing Check the following Baseline Multicomponent Heavy Smooth Option Peak detection B 150 G 150 Y 150 R 150 Size Call Range Min 75 Max 400 Size Calling Local Southern Method Split Peak Correction Check none Note It may be necessary to adjust the range of data points analyzed in the Analysis Range section of the Analysis Parameters window in order to capture all sizes from 75 bp to 400 bp In addition database profiles are derived from single source samples At the analyst s discretion a Peak Amplitude Threshold of 50 RFUs may be used for low level alleles The Technical Leader must review and approve any all
232. ntrator add 100 ul Transfer the aqueous phase from the tube in Step E to the concentrator Avoid pipetting organic solvent from the tube into the concentrator G Place a cap on the concentrator and spin in a microcentrifuge at 2500 x g for 10 minutes H Add 100 ul TE to the concentrator Replace the cap and spin the assembly microcentrifuge at 2500 x g for 10 minutes l Remove the cap and add a measured volume of that is between 20 ul and 200 ul to the concentrator Remove the concentrator from the filtrate cup and carefully invert the concentrator into a labeled retentate cup Discard the filtrate cup Note Final recovery volumes following purification generally range from 20 ul for evidentiary question samples to 200 ul for reference samples known samples The final recovery volumes for the extraction reagent blanks for evidentiary samples and known samples are generally 100 and 200 ul respectively Page 41 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 Spin the assembly in a microcentrifuge at 2500 x g for 5 minutes Discard the concentrator Cap the retentate cup Estimate the quantity of DNA in the sample by slot blot hybridization After quantification the samples can be amplified es Store samples at 2 8 C or frozen Prior to the use of samples after storage they should be vortexed briefly and spun in a microcentrifuge for
233. nvestigation of Species Specificity Using Nine PCR Based Human STR Systems J For Sci 40 1995 952 6 DNA recommendations 1994 Report Concerning Further Recommendations of the DNA Commission of the ISFH Regarding PCR based Polymorphisms in STR Short Tandem Repeat Systems Editorial For Sci Intl 69 1994 103 4 Page 192 of 255 Appendix C Resources CBI DNA Analysis SOP Vers 2 1 06 13 02 4 43 PM CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 Edwards A Civitello Hammond and Caskey DNA Typing and Genetic Mapping with Trimeric and Tetrameric Tandem Repeats Am J Hum Genet 49 1991 746 56 Fowler J C S Burgoyne L A Scott A C and Harding H W J Repetitive Deoxyrobonucleic Acid DNA and Human Genome Variation A Concise Review Relevant to Forensic Biology J For Sci 33 1988 1111 26 Fregeau C J and Fourney R M DNA Typing with Fluorescently Tagged Short Tandem Repeats A Sensitive and Accurate Approach to Human Identification BioTechniques 15 1993 100 19 GeneAmp PCR System 9700 User s Manual Set 1997 Guidelines for Operation of the Combined DNA Index CODIS Laboratory Aug 1997 Guttman A and Cooke N Effect of Temperature on the Separation of DNA Restriction Fragments in Capillary Gel Electophoresis Journal of Chromatography 559 1991 285 94 Isenberg A R McCord B R Koons B W Budl
234. o NDIS on a monthly basis maintaining user information and informing the FBI of any changes 8 responding to any and all requests for information from the FBI completing paperwork for putting new laboratories on the system 10 keeping abreast of any software or hardware recommendations 11 training of new analysts and Local CODIS Administrators 12 notifying the CBI Deputy Director of NDIS hits D The State CODIS Administrator has jurisdiction over all CODIS laboratories in the State of Colorado and has the authority to terminate any laboratory s participation in CODIS in the event of a problem until the reliability of the computer data can be assured DNA PROFILE MANAGEMENT APPROVED DNA PROFILES FOR LDIS Samples to be entered into LDIS must have been obtained while following all of the NDIS Standards for Acceptance of DNA Data These DNA profiles must be obtained using a validated standard operating procedure that utilizes an NDIS accepted PCR kit The positive control must have been typed correctly and there must be no signs of contamination For forensic profiles only the alleles that are attributed to the putative perpetrator s may be entered into LDIS Alleles derived from forensic profiles that are unambiguously attributed to a victim or individuals other than the perpetrator s such as but not limited to a husband or boyfriend SHALL NOT be entered into LDIS DNA profiles from convicted offenders must be obtained from standards
235. o precautions concerning having multiple tube lids open simultaneously and changing pipette tips between samples are relaxed for this extraction procedure In addition since a large amount of input DNA is bound to the punch the amplification cycle number may be reduced A B Using a clean 1 2 mm Harris punch remove a sample from the stain and place it into an amplification tube Add 200 ul FTA Purification Reagent to each tube Cap each tube and vortex 1 to 2 seconds at slow speed Allow the tubes to sit for 5 minutes at room temperature with a second brief vortex halfway through the incubation After the 5 minute incubation vortex for a third time and then carefully remove as much of the reagent as possible Repeat Steps C and D an additional two times for a total of three washes with the FTA Purification Reagent After the FTA Purification Reagent has been removed for the third time add 200 ul 10mM Tris HCl pH 8 0 0 1mM EDTA Cap each tube and vortex 1 to 2 seconds at low speed Allow the tubes to sit for 5 minutes at room temperature with a brief vortex halfway through the incubation Remove the and replace with an additional 200 ul TE Cap each tube and vortex 1 to 2 seconds at low speed Allow the tubes to sit for 5 minutes at room temperature with a brief vortex halfway through the incubation Remove the TE and allow the FTA paper punch to air dry Alternatively go directly to Step K Add TE to th
236. o the HLA DQA1 1 2 1 3 and 4 1 alleles The All but 1 3 dot is positive in the presence of all HLA DQA1 alleles EXCEPT 1 3 This probe is necessary to differentiate the 1 2 1 3 genotype from the 1 3 1 3 genotype NOTE The All but 1 3 dot can be equal to or lighter than the C dot when the genotype has an HLA DQA1 1 3 allele paired with an HLA DQA1 4 1 4 2 or 4 3 allele because the HLA 4 1 4 2 4 3 alleles have single partially destabilizing mismatch to the but 1 3 The partially destabilizing mismatch allows these three alleles to bind to this probe weakly relative to the HLA DQA1 1 1 1 2 2 and 3 alleles Two additional HLA DQA1 sub typing probes differentiate the HLA DQA1 4 1 allele from the HLA DQA1 4 2 and 4 3 alleles The 4 1 dot is positive only in the presence of the HLA DQA1 4 1 allele The 4 2 4 3 dot is positive in the presence of HLA DQA1 4 2 and 4 3 alleles An example of a developed AmpliType HLA DQA1 DNA Probe Strip using PCR product amplified from 2 ng of Control DNA 1 is shown in Figure 3 The AmpliType HLA DQA 1 type for Control DNA 1 is 1 1 4 1 Page 81 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 1 2 4 2 1 234 u O41 O43 DQA1 4 1 3 Figure 3 The AmpliType HLA DQA1 type for Control DNA 1 PERFORMANCE CHARACTERISTICS The AmpliType PM and AmpliType PM
237. o the concentrator Avoid pipetting organic solvent from the tube into the concentrator Place a cap on the concentrator and spin in a microcentrifuge at 2500 x g for 10 minutes Add 100 ul to the concentrator Replace the spin cap and spin the assembly in a microcentrifuge at 2500 x g for 10 minutes Remove the cap and add a measured volume of that is between 20 ul and 200 ul to the concentrator Remove the concentrator from the filtrate cup and carefully invert the concentrator onto a labeled retentate cup Discard the filtrate Note Final recovery volumes following purification generally range from 20 ul to 200 ul for evidentiary samples The final recovery volumes for the extraction reagent blanks for evidentiary samples are generally between 20 ul and 200 ul Spin the assembly in a microcentrifuge at 2500 x g for 5 minutes Discard the concentrator Cap the retentate cup Estimate the quantity of DNA in the sample by slot blot hybridization After quantification the sample can be amplified Store samples at 4 C or frozen Prior to the use of samples after storage they should be vortexed briefly and spun in a microcentrifuge for 5 seconds SLIDE MOUNTED HAIR SPECIMENS A Loosen the slide coverslip by carefully pipetting xylene around the coverslip edges If the coverslip does not loosen the entire slide can be placed into a Petri dish and covered with xylene for one or more hours until the coverslip has loo
238. o whether or not the sample will be analyzed If a blood tube is broken or leaking at the time of submission to CBI the sample will not be accepted Preparation of Stain Card From Liquid Blood A A stain card will be prepared for each blood sample Each stain card will contain the offenders name agency case number CBI case number and a unique PCR number assigned by CBI personnel Preparation of bloodstain cards will be carried out in a manner to minimize risk of contamination T Handle the cards wearing gloves Change gloves between samples when spotting the blood sample or when contact with blood may have occurred 2 Prepare each new stain card on a clean surface Use a new tube decapper or fresh Kimwipe for each blood sample Do not allow prepared stain cards to come in direct contact with each other or with unprepared cards Upon completion of preparing the stain card the offenders blood tube and any remaining blood will be sterilized by autoclaving followed by disposal in the biohazard trash When the spotted stain card is thoroughly dry the card will be packaged in a plastic sleeve heat sealed with initials and date and stored appropriately The FTA Cards are stored locked at room temperature The S amp S cards are stored in a locked 70 C freezer Storage of Buccal Samples Buccal samples on cotton swabs are stored in the 70 freezer Buccal samples on FTA cards are stored locked at room temperatur
239. of birth agency case number offense code and signature are required If any required information is not present the submitting agency is notified and the information needed is obtained All data is entered into the stand alone DNA database computer Every time data is entered into the computer a back up disk is created Periodically a hard copy of recent submissions is generated and used to record which samples have been analyzed by whom and which remaining samples require testing This printout is stored in the STR Binder located in the Denver DNA prep area B Preparation Of Stain Card From Liquid Blood 1 A stain card is prepared for each blood sample Each stain card contains the offenders name agency case number case number and a unique confidential PCR number assigned by CBI personnel Preparation of FTA blood stain cards is carried out in a manner to minimize risk of contamination The precautions include a All blood stain cards are prepared in a hood The individual preparing the card should wear protective eyewear and clothing b The FTA cards are handled wearing gloves Gloves are changed when contact with blood may have occurred Each new stain card is prepared clean surface d A fresh lab tissue is used to open each EDTA blood tube e Each FTA card is spotted with a fresh sterile transfer pipet One to two drops of blood is placed within the circle of the card f Prepared stain cards m
240. of hits regarding their cases j ensuring that both the State Administrator and CBI Deputy Director are informed of hits within their laboratories The Local CODIS Administrator has the authority to terminate the laboratory s participation in CODIS in the event of a problem until the reliability of the computer data can be assured STATE SDIS CODIS ADMINISTRATOR A The State CODIS Administrator along with the Technical Leader is responsible for ensuring that the DNA sections in all participating local CODIS laboratories are in compliance with all aspects of the NDIS Standards for Acceptance of DNA Data including quality assurance of the data The State CODIS Administrator must satisfy all duty requirements responsibilities of the Local CODIS Administrator and functions as the Local CODIS Administrator for the Denver laboratory Additional responsibilities of the State CODIS Administrator include but are not limited to 1 processing the LDIS to SDIS uploads from each local laboratory 2 routine autosearches 3 processing remote search requests 4 sending the SDIS to NDIS upload weekly 5 processing outside agency search requests Page 165 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 6 obtaining the local laboratories hit records maintaining hit records for the CBI Denver local laboratory and forwarding hit counts for the State of Colorado t
241. on 12 JUNE 2002 POSSIBLE CAUSE The test sample contains PCR inhibitor Input DNA and or PCR product was not denatured sufficiently during amplifi cation Amplified DNA was not denatured Hybridization or Stringent Wash temperature too high or too low Hybridization or PM DQA1 Wash Solution salt concentration too high or too low Stringent Wash time too long or too short Mixed sample or contami nation Page 87 of 255 CBI DNA Analysis SOP Vers 2 1 RECOMMENDED ACTION See AmpliType User Guide Version 2 Section C for notes on DNA sample preparation Check calibration of the GeneAmp PCR Instrument System Temperature Verifi cation System Check GeneAmp PCR Instrument System block temperature 95 C leave sample in block gt 3 minutes complete DNA addition to tray within 20 seconds repeat test Check that rotating water bath temperature is 55 C 1 C repeat test Prepare new _ solutions repeat test Repeat test washing for 12 minutes 1 minute See References 14 15 17 18 and 28 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 OBSERVATION 10 Weak or absent 4 1 dot on the AmpliType HLA DQA1 DNA Probe Strip in the amplified Control DNA 1 sample 11 41 2 1 3 4 weaker than dot on AmpliType HLA DQA1 DNA Probe Strip 12 1 1 dot weaker than dot but no signal for t Ampli
242. orrect size calling of the GS internal size standard assigned by GeneScan Analysis software D Execute Kazam 20 filter macro When finished a plot window opens containing the labeled alleles for the blue data The analyst may wish to close this Page 150 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 plot window and sort the data at this point This can be done by selecting View Dye lane sorting NOTE The first sample file containing an allelic ladder that is found in the Dye Lane list is the one that is used by the macro to determine the size of the allele categories that will be used for genotyping If you use a ladder other than the first ladder then you must remove the ladder designation from the Dye Lane list Simple removal of the will suffice E Examine data by looking through sorted data or by examining by individual color EXAMINING DATA A Insure that the peaks of the allelic ladder are labeled with the correct allele designations Peak labeling criteria are defined by the GT template being used Allele categories and their designations can be viewed in Genotyper under View Categories Peaks that do not size within an allele category will have a label indicating OL Allele Off Ladder Allele B Peaks less than the minimum peak height previously specified in GeneScan software should not be labeled A frequently encountered GenoTyper anomaly ha
243. ose Save As from the File menu to save the DataBankSTR file Transfer the DAT to CODIS computer by way of the shared folder on the CODIS server ANALYST ACTIVITY An analyst activity form is filled out for each sample Include the following LAB the CBI case number SEC DBS ANA analysts initials DOS date of submission TP1 DBS SB1 DOC PRO DYC or COM SP1 1 TRV reviewing analyst s initials Page 159 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 NAMING CONVENTION FOR STR ANALYSIS OF DATABASE SAMPLES NOTE The date the samples were run on instrumentation is the basis for naming electronic files Date mm dd yy month day year Sample Number MACINTOSH FILE NAMES Run Folder Reviewed Run Folder Genotyper file Reviewed file CODIS ready Genotyper file Genotyper table Reviewed table CODIS ready Genotyper table DataBankSTR file DataBankSTR CMF file WINDOWS NT FILE NAMES CODIS ready Genotyper file Reviewed file Genotyper table Reviewed table CMF Conversion Report Excel CMF file CREATING CDs PCR number CBI case number Ex 10007005555 Run Folder mm dd yy anaPP CO Run Folder mm dd yy anaPP COrev GT mm dd yy anaPP CO GT mm dd yy anaPP COrev CODIS GT mm dd yy anaPP CO GT mm dd yy anaPP CO GT mm dd yy anaPP COrev CODIS GT mm dd yy anaPP CO mmddyyan mmddyyan DAT CODISGTdateanalD CODISGTdateanalDana2 GTdatean
244. ot on the same strip JUNE 2002 POSSIBLE CAUSE Insufficient water washes after Color Development Cross hybridization caused by Hybridization or Stringent Wash tem perature being too low Cross hybridization caused by Hybridization or PM DQA1 Wash Solution salt concentra tion being too high Cross hybridization caused by Stringent Wash time being too short Contamination by ampli fied product or samples Hybridization or Stringent Wash temperature too high or too low Hybridization or PM DQA1 Wash Solution salt concentration too high or low Stringent wash time too long or short Mixed sample or contami nation Page 85 of 255 CBI DNA Analysis SOP Vers 2 1 RECOMMENDED ACTION Increase number of water washes in future assays Check that rotating water bath temperature is 55 C 1 C repeat test Prepare new solutions repeat test Repeat test washing for 12 minutes 1minute See AmpliType User Guide and References 18 and 28 Check that rotating water bath temperature is 55 C 1 C repeat test Prepare new solutions repeat test Repeat test washing for 12 minutes 1 minute See User Guide References 14 15 17 18 and 28 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 OBSERVATION 7 More than two alleles present on the AmpliType HLA DQA1 DNA Probe Strip or at the HBGG and or GC marker on the Amp
245. ount of amplified DNA or control sample to each tube Sample dilution for loading is dependent on extraction method comb used individual sample and instrument The proper dilution needs to be determined by each analyst as a part of his or her validation studies 8 A minimum of two ladders should be run on each gel 9 Vortex and spin each sample tube The remaining sample preparations steps should be performed during the PreRun Step J below 10 Denature samples at 95 C for a minimum of 5 minutes and no longer than 10 minutes Page 136 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 11 Snap cool on ice water bath immediately Samples must remain on ice until loading Choose NEW from the File menu of the 377XL Collection software Click on GeneScan Run Import a sample sheet by clicking on the arrows in the Sample Sheet box and selecting the appropriate sample sheet Set Auto Analysis parameters Check that comb type and number of lanes settings are correct Set up the gel on the instrument T Place the lower buffer chamber on the 377 and plug in the electrode 2 Remove the clamps from the gel 3 Thoroughly clean excess acrylamide from the glass plates using ultrapure water and Kimwipes 4 Carefully remove the casting comb and clean the comb area Remove all excess acrylamide Insert a single use sharkstooth comb approximately 1 8 into the acrylamide U
246. owe B and Allen R O DNA Typing of a Polymerase Chain Reaction Amplified D1S80 Amelogenin Multiplex Using Capillary Electrophoresis and a Mixed Entagle Polymer Matrix Electrophoresis 17 1996 1505 11 Issaq H Chan K Mischik G The Effect of Column Length Applied Voltage Gel Type and Concentration on the Capillary Electrophoresis Separation of DNA Fragments and Polymerase Chain Reaction Products Electrophoresis 18 1997 1153 8 Lincoln P ed DNA Recommendations Further Report of the DNA Commission of the ISFH Regarding the Use of Short Tandem Repeat Systems For Sci Intl 87 1997 179 84 Kimpton C P Walton A and Gill P A Further Tetranucleotide Repeat Polymorphism in the vWF Gene Hum Mol Genet 1 1992 287 Kimpton C P Gill P Walton A Urquhart A Millican E and Adams M Automated DNA Profiling Employing Multiplex Amplification of Short Tandem Repeat Loci PCR Methods and Applications 3 1993 13 22 Kline M Duewer D P Redman J Reeder D and Richard Interlaboratory Evaluation of Short Tandem Repeat Triplex CTT J For Sci 42 1997 897 906 Page 193 of 255 Appendix C Resources CBI DNA Analysis SOP Vers 2 1 06 13 02 4 43 PM CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 Lazaruk K Walsh Pl Oaks F Gilbert D Rosenblum Menchen S Scheibler D Wenz H Holt C and Wallin J Genotyping of Fore
247. peat Polymorphism at 021511 locus Hum Mol Gent 1 1 1992 67 Siles B Collier G B Reeder D J May W E The Use of New Gel Matrix for the Separation of DNA Fragments a Comparison Study Between Slab Gel Electrophoresis and Capillary Electrophoresis Applied and Theoretical Electrophoresis 6 1996 15 22 STR Analysis ABI 310 Genetic Analyzer Protocol Armed Forces DNA Identification Laboratory Nov 1998 Sullivan K M Pope S Gill P and Robertson J Automated DNA Profiling by Fluorescent Labeling of PCR Products The Forensic Science Service Case Report No 742 1992 1 20 Page 194 of 255 Appendix C Resources CBI DNA Analysis SOP Vers 2 1 06 13 02 4 43 PM CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 Urquhart A Kimpton C P Downes T J and Gill P Variation in Short Tandem Repeat Sequences a Survey of Twelve Microsatellite Loci for Use as Forensic Identification Markers Int J Leg Med 107 1994 13 20 VanOorschot R Gutowski S Robinson S Hedley J and Andrew 1 1 Validation Studies Effect of Substrate Environment and Mixtures J For Sci 41 1996 142 5 Wallin J M Buoncristiani M R Lazaruk K D Fildes N Holt C L and Walsh P S TWGDAM Validation of the AmpF STR Blue PCR Amplification Kit for Forensic Casework Analysis J For Sci 43 4 1998 117 Walsh P S Fildes N J
248. performed prior to the purification steps Assemble a Microcon concentrator unit To the top of the concentrator add 100 ul Transfer the aqueous phase from the tube in Step L to the top of the concentrator Avoid pipetting organic solvent from the tube into the concentrator Place a cap on the concentrator unit from the assembly and spin in a microcentrifuge at 2500 x g for 10 minutes Add 100 ul to the concentrator Replace the cap and spin the assembly in a microcentrifuge at 2500 x g for 10 minutes Remove the cap and add a measured volume of that is between 20 ul and 200 ul to the concentrator Remove the concentrator from the filtrate cup and carefully invert the concentrator onto a labeled retentate cup Discard the filtrate cup Note Final recovery volumes following purification generally range from 20 ul to 200 ul for evidentiary samples The final recovery volumes for the extraction reagent blanks for evidentiary samples are generally between 20 ul and 200 ul Spin the assembly in a microcentrifuge at 2500 x g for 5 minutes Discard the concentrator Cap the retentate cup Estimate the quantity of DNA in the sample by slot blot hybridization After quantification the sample can be amplified Store samples at 4 C or frozen Prior to the use of samples after storage they should be vortexed briefly and spun in a microcentrifuge for 5 seconds Page 43 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FO
249. pment become contaminated comparable cleaning procedures should be followed Page 14 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 EVIDENCE HANDLING A clear well documented chain of custody is maintained from the time the casework evidence is first received at the CBI Laboratory until it is released from the Laboratory to the submitting agency Each sample is labeled with a unique identifier in accordance with CBI policy This includes all of the extracted DNA samples All evidence and samples from evidence are to be received collected handled sampled and stored so as to preserve the identity integrity condition and security of the item After a DNA analysis has been completed the samples of amplified DNA and the amplifications of corresponding control samples are discarded in the PCR amplification and typing room s Strips are photographed and dried on non absorbent film i e sheet protectors The strips photographs or copies thereof are kept in the specific case file The microcentrifuge tubes containing the extracted DNA are placed into heat sealed plastic bags and then placed in the DNA packet along with the remaining stain cards swabs cuttings samples and controls The DNA packet is stored in the evidence freezer at the CBI Laboratory until it is returned to the submitting agency A well documented chain of custody is also maintained for database samples The re
250. r loci in the thermal cyclers Turn on the thermal cycler and allow the instrument to warm up for a minimum of 1 hour If using the 480 add one drop of oil into each of the wells to be used Select the file for forensic STR analysis The parameters are listed below Page 130 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 DNA THERMAL CYCLER 480 9700 Initial Incubation 1 cycle 95 C 11 minutes Cycle step cycle file 25 28 cycles 94 C 1 minute 59 C 1 minute 72 C 1 minute Final extension 1 cycle 60 C 90 minutes Hold temperature soak file 10 C indefinite Tubes can be left in the thermal cycler at 4 10 C until removed The amplified samples can then be stored at 2 6 C for a week or longer at 15 to 20 C Blood spotted on FTA paper is typically amplified at 25 cycles this cycle number is determined through validation and may be dependent on the lot of FTA paper Samples spotted on other substrates are typically amplified at 28 cycles Some samples due to their age and condition may require an extra final extension step of 30 45 minutes to reduce typing AMPLIFICATION FORMULATIONS The following formulation applies to both the Profiler Plus and Cofiler kits A D E F Vortex the PCR reaction mix primer set and AmpliTaq Gold DNA polymerase supplied in the kit for 5 seconds at medium speed Spin the tubes briefly in a mi
251. r the possible presence of contamination occurring during the amplification setup If a negative control exhibits peaks gt 50 RFU not attributable to artifacts located between 100 and 350 bp or at 75 bp and 400 bp they indicate the presence of contamination and none of the samples amplified with the negative amplification blank will be considered inclusive for match purposes If they cannot be re amplified then these samples can be considered for exclusion purposes If artifactual peaks occur then the negative amplification blank must be re injected POSITIVE EXTRACTION AND AMPLIFICATION CONTROLS The positive control should have all the alleles present and they must be genotyped correctly If there appears to be a problem with the injection or an electrophoretic Page 115 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 problem the control and ladder can be re injected If the correct type is obtained the run can be used for match purposes If this does not solve the problem the entire set should be re amplified and typed The previous run can be considered for exclusion purposes but will be inconclusive for match purposes INTERPRETATION OF THE SPECIMENS The following interpretation standards are to be used as a guide This set of guides will cover most routine case scenarios Cases that fall outside of these guidelines will be addressed through discussion with the Technical R
252. rations should be performed when the instrument is being installed has recently been serviced in an area that affects the laser or CCD camera optics or if the baseline in the data cannot be corrected by the current spectral calibration Follow the procedure in the ABI PRISM 3100 Genetic Analyzer Users Manual in Chapter 4 that different spectral calibrations are used and are determined by the type of genetic profiling kit used in the analysis Plates and plate records There are six methods for creating plate records defined in the ABI 3100 User s Manual The Colorado Bureau of Investigation DNA Database Section has created an Excel text format import file of a 96 well plate layout called 3100 Load Sheet and Plate Record The plate record created is exported as a tab delimited text file format and is imported onto the 3100 Collection software as such The plate layout may be printed out for use in loading the PCR product into the 96 well plate It is saved as an Excel file An additional option is to create a plate record on the instrument using the 3100 collection software The plate record has several fields that must be filled Well Position Sample Name Dye Set Color Number Standard Dye Color Info Color Comment BioLIMS Project Run Module and Analysis Module The following procedure applies to both methods 1 Fill in the Sample Name and Color Info sections with the CBI sample name Double check the CBI sample names
253. rator from the filtrate cup carefully invert the concentrator onto a labeled retentate cup Discard the filtrate cup Note Final recovery volumes following purification generally range from 20 ul to 200 ul for evidentiary samples The final recovery volumes for the extraction reagent blanks for evidentiary samples are generally between 20 ul 200 ul Page 47 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 Spin the assembly in a microcentrifuge at 2500 x g for 5 minutes Discard the concentrator Cap the retentate cup Estimate the quantity of DNA in the sample by slot blot hybridization After quantification the sample can be amplified Store samples at 4 C or frozen Prior to the use of samples after storage they should be vortexed briefly and spin in a microcentrifuge for 5 seconds HAIRS UNMOUNTED HAIR SPECIMENS A Handling hair with clean forceps examine the hair under a dissecting microscope for the presence of sheath material The hair may be placed on a clean piece of white paper Note possible presence of body fluids on hair B Wash the hair containing sheath material thoroughly to reduce surface dirt and contaminants by immersing the hair in sterile saline Follow this by either of the following cutting methods 1 Rinse thoroughly in ethanol and place onto clean paper Use a clean scalpel or clean scissors to cut a 1 cm portion
254. re amplification can lead to results that are not interpretable Do NOT bring amplified DNA or equipment and supplies used to handle amplified DNA into the DNA extraction PCR setup area NOTE Wear clean disposable laboratory gloves while preparing samples for PCR amplification Change gloves frequently or whenever there is a chance they have been contaminated with DNA Page 69 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 Turn on the GeneAmp PCR Instrument System Thermal Cycler 480 and program as follows see Instrument Manual File 30 32 Cycles Melt 1 minute 94 C Anneal 30 seconds 60 C Extend 30 seconds 72 C File 10 Final Step 7 minutes 72 C File 20 Soak indefinite 15 C NOTE These programs may be saved as User Files for later use see Instrument Manual As with any calibrated piece of laboratory equipment the GeneAmp PCR Instrument System should have a documented temperature verification test performed semiannually Prepare the DNA test samples for addition to the PCR amplification reactions Each DNA sample shall be quantitated with the QuantiBlot Human DNA Quantitation Kit Part No N808 0114 The final DNA concentration should be in the range of 0 1 to 0 5 so that 2 to 10 ng of DNA will be added to the PCR reaction in a volume of 18 ul If the sample DNA concentration is greater than 0 5 dilute a portion of
255. re testing will be performed Investigating officers will be notified Reports will be issued to reflect the notification of the officers of the hit A copy of the CODIS match report will be placed into each of the case files Copies of the case report STR allele worksheet the case submittal sheet and any written correspondence regarding the hit will be sent to the State Administrator The hit will be recorded on the CODIS hits worksheet as well as the hit scorecard If a possible hit is made as a result of an outside agency search request fax request or keyboard search 1 The analyst involved in the case will be notified so that the DNA profiles can be reviewed The outside agency will be notified of the potential match by telephone or letter The results will be discussed The analyst will determine along with the outside agency analyst if further DNA analysis is required The analyst s course of action will be documented in the case file If the hit results in an investigative lead the identifying information for the DNA profile will by forwarded to the outside agency It is the responsibility of the agency requesting the search to notify the investigating officers The CODIS Administrator will keep copies of the match report and any other documentation The hit will be classified using the CODIS hit counting guidelines The NDIS Custodian will be notified of any interstate hits in writing per NDIS procedure
256. rensic Unknown Database The Unidentified Human Remains Database and the Relatives of Missing Person Database are not currently in use at CBI Profiles are entered by the local laboratory into the appropriate LDIS database The local systems upload profiles to SDIS in the CBI Denver lab The state level is then responsible for uploading the collected profiles to the national level At CBI each of the three laboratories analyzes and enters DNA profiles from both forensic evidence and convicted offenders Forensic unknown DNA profiles may be searched against other forensic unknown profiles and convicted offender profiles in that local system These searches may result in local case to case links or generate investigative leads in the event of a hit against the Convicted Offender Database Upon upload to the state system those DNA profiles will be searched against forensic unknown profiles from other local laboratories as well as all persons contained in the statewide Convicted Offender Database Profiles from convicted offenders containing all 13 CODIS core loci and profiles from forensic unknown samples with a minimum of 10 of the CODIS core loci are acceptable for upload into NDIS These profiles will be compared to the forensic unknown profiles and convicted offender profiles from other states contained within NDIS Page 163 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 Forensic DNA la
257. reparation Dissolve 100 mg Proteinase K in 10 ml sterile ultrapure water Aliquot solution 0 5 ml recommended and store frozen at 20 C Date Verified by Prepared Amount Proteinase K lot Preparer initials date PROK Page 242 of 255 Appendix Al DNA Working Solutions Log Proteinase K 10 mg ml CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 A APPENDIX AJ DNA WORKING SOLUTIONS LOG QUANTIBLOT PREWETTING SOLUTION MODEL 0 4N NAOH 25MM EDTA 500 ML Solution Preparation Add 20 10N NaOH 25 0 5M EDTA to 455 ml ultrapure water and mix thoroughly Date Prepared Amount 10 N NaOH 0 5M EDTA Preparer lot lot QBPREWET Page 243 of 255 Appendix AJ DNA Working Solutions Log QuantiBlot PreWetting Solution CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 APPENDIX AK DNA WORKING SOLUTIONS Loc QUANTIBLOT SPOTTING SOLUTION MODEL 0 4N NAOH 25MM EDTA 0 00008 BROMOTHYMOL BLUE 75 ML Solution Preparation Add 3ml 10N NaOH 3 75 ml 0 5M EDTA and 150 uL 0 04 Bromothymol Blue provided in kit to 68 ml ultrapure water and mix thoroughly Note Spotting solution is stable for at least three months at room temperature 10N NaOH 0 5M EDTA Date Prepared AMOUNT lot lot
258. rifuge Place the appropriate number of sample tubes in the tray and label appropriately Add to each tube 25 ul of the formamide ROX mixture Following the Sample Sheet format add samples as follows a Cap a tube with septum BLANK b Add to two tubes 1 5 ul of the appropriate allelic ladder and cap with septum Add 1 5 ul of each sample into the appropriate tube and cap with septum before going to the next tube NOTE Septa or Septa Strips melt at high temperatures Do not close the lid of the 9700 Thermal Cycler when denaturing the septa will adhere to the lid Do not autoclave or re use septa Make sure that samples are mixed thoroughly and without bubbles on the bottom of the tube Vortex each sample and spin in a centrifuge Denature the samples at 95 C for 3 minutes no more than 5 minutes Snap cool on ice water bath 4 C immediately for a minimum of 3 minutes Secure the septa Replace samples in the tray Under the Manual Control window select Present Autosampler followed by Execute Open the door and place tray onto the Autosampler with position A1 at the upper right hand corner Return tray to position Manual Control Autosampler Retum Execute Close door Prepare notes sheet Include the following information as a minimum 1 2 3 4 Instrument ID Capillary lot Buffer lot POP 4 lot Page 107 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY
259. rited these alleles from their biological parents If a suspected parent and a child share an obligate allele then the individual cannot be excluded as a potential biological parent The frequency of other Random Parents Not Excluded RPNE in the population would then need to be calculated This frequency the RPNE determines the number of random individuals that could also not be excluded as potential biological parent s This frequency is much the same as what is calculated for single source samples The question being answered is what is the chance that a random person would also not be excluded as a potential biological parent The RPNE is based on the obligate alleles in the child s sample The calculations are as follows If at locus 1 the child is heterozygote has two obligate alleles then any person with either of those obligate alleles could not be excluded as a potential biological parent So for example if at locus 1 the child is type 8 10 then any individual with an 8 or a 10 at locus 1 could not be excluded Therefore the RPNE is going to equal the frequency of 8 8 10 10 8 10 8 X and 10 X or RPNE locus 1 q 2pq 2 1 2q 1 p q 1 1 4 equation 1 If on the other hand the child is homozygote has one obligate allele then any person with that allele could not be excluded as a potential biological parent So for example if at locus 2 the child is type 15 15 then any individual with a 15 at locus
260. s If a possible hit occurs at the National level NDIS interstate hits 1 The analyst involved in the case will be notified so that the DNA profiles can be reviewed Page 172 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 2 The NDIS Procedures Manual lists specific instructions for contacting CODIS laboratories issuing reports and notifying the NDIS Custodian in the event of an interstate hit The CBI will follow NDIS procedures in the event of an NDIS hit 3 It is the responsibility of the CODIS laboratory that submitted the matching profile to notify the investigating officers of the hit and to provide them with contact information for the appropriate out of state agency 4 The hit will be classified using the CODIS hit counting guidelines In the event of a hit against a profile in the Convicted Offender Database the identification information for that sample may be verbally released to the appropriate agency or investigator s prior to re analysis of the sample The investigating officer s or agency should be informed that the hit is not confirmed and that the laboratory will contact them once the confirmation process has been completed The identification information for the Convicted Offender Database is contained in a file on a stand alone computer in the Denver laboratory In the event of a hit against the Convicted Offender Database the identification in
261. s D The temperature verification kit is sent out annually for recalibration Each thermal cycler used for amplification is verified with a NIST traceable standard annually HYBRIDIZATION BATHS AND HEATING BLOCKS A The hot shaker water bath s and heating block s temperatures are checked prior to use and monitored during use with a NIST traceable thermometer B The NIST traceable thermometer is sent out for recalibration annually PIPETTORS Pipettors are sent to a contractor annually for cleaning calibration and general maintenance CENTRIFUGES Centrifuges are cleaned and serviced as necessary pH METER The pH meter is calibrated prior to each use with the appropriate buffer s of known pH BALANCES Balances are serviced and recalibrated annually according to service contract specifications They are checked in house monthly by weighing known standard weights and comparing the displayed value to the known value Page 19 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 WATER PURIFICATION SYSTEMS Water purification systems are maintained and are sanitized as needed REFRIGERATORS FREEZERS 20 C AND 70 C AND INCUBATORS Refrigerators freezers 20 C and 70 C and incubators are maintained and temperatures are taken on a weekly basis Temperatures are logged on a temperature log sheet specific for each piece of equipment ABI 377 ABI 310
262. s been the labeling of peaks that are below GeneScan thresholds These should be manually deleted and noted by the analyst C Scroll through the samples to examine the peak labels and edit the peak labels where necessary Clicking on a labeled peak will remove the label Clicking on the same peak again will label the peak by size bp Samples that do not meet standards may be removed by opening the dye lanes window highlighting the sample name and selecting Clear under the Edit menu Note To change a label choose Change label from the Analysis menu and select the desired label D Once the samples have been edited create an allelic table by executing the Make Allele Table macro Keep a print out of the allele table with the sample records To print export the table then open it in Excel Save the project before proceeding i e GT dateanalD PP CO As part of the technical review process second reviewing analyst should follow the above steps in the Examining Data section saving files with his her initials added at the end i e GT mm dd yy anaPP COrev F Once the typing is confirmed by a technical review the DNA profiles may be imported into CODIS See following section on how data is confirmed Page 151 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 INTERPRETATION OF DATA INTRODUCTION The interpretation of database profiles is a matter of both prof
263. s into CODIS SAMPLE INPUT PROTOCOL DATABASE A Technically reviewed database profiles may be imported into CODIS as CMF common message format files and transferred into LDIS using the batch transfer function Each analyst is responsible for transferring his her own data into LDIS Alternatively individual profiles may be entered using the keyboard entry procedure described for casework profiles the Convicted Offender Specimen Category should be selected B The following Specimen ID format shall be used for database samples PCR Case i e 10007000000 There are to be no spaces or punctuation C Only DNA Analysts having passed the FBI background check will enter profiles into CODIS D The following is the procedure for importing CMF files into LDAS 1 Log on to CODIS Import by entering your CODIS User ID and Password 2 Select a file to import a Click on the file open icon b Select the proper CMF file Click on OK 3 Validate the selected file a Click on the validate icon CODIS will perform the validation 4 Examine the Import Status results a Click on the printer icon b Choose screen C The action taken for a particular profile will be described red message indicates problems with the data d Close the Import Status results file 5 Import the CMF file a Click on the import arrow icon CODIS will import the profiles Page 169 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATO
264. s of sperm K Spin in a microcentrifuge for 5 minutes at 10 000 to 15 000 x g L Add 150 ul of the supernatant to 50 ul of 20 Chelex in a fresh 1 5 ml microcentrifuge tube Save for epithelial DNA analysis beginning with Step S M Wash the sperm pellet as follows Re suspend the pellet in 0 5 ml DIGEST BUFFER Vortex briefly Spin in a microcentrifuge for 5 minutes at 10 000 to 15 000 x 9 Remove all but 50 ul of the supernatant and discard the supernatant N Repeat wash Step M an additional 2 times NOTE Additional wash steps are recommended when the ratio of sperm to epithelial cells is low O Wash once with sterile distilled water as follows Re suspend the pellet in 1ml sterile water Vortex briefly Spin in a microcentrifuge for 5 minutes at 10 000 to 15 000 x g Remove all but about 50 yl of the supernatant and discard the supernatant P Re suspend the pellet by stirring with a sterile pipette tip Remove about 3 ul of the re suspended sample and spot on a glass microscope slide for cell examination Stain with Christmas Tree Stain refer to the CB Forensic Laboratory Serology SOP Staining Of Spermatozoa Kernechtrot Picroindigocarmine Steps to K M to P may be repeated if nucleated or non nucleated non sperm cells are detected in the cell examination Q Add about 150 ul of 5 Chelex to the approximately 50 ul re suspended sperm cell pellet final volume should be about 200 ul Add 2 ul of 10 mg ml Proteinas
265. s to the PCR amplification and typing area is restricted to DNA analysts and supervisors Production and handling of amplified DNA is restricted to the PCR amplification and typing rooms so that strict physical isolation of PCR product is maintained to avoid transfer of amplified DNA into preparatory areas DNA amplification DNA typing hybridization stringent wash color development gel electrophoresis and capillary electrophoresis and waste disposal of amplified DNA solutions are carried out in this area All equipment glassware and supplies within this room are dedicated to PCR amplification and typing Equipment and supplies used to handle amplified DNA are not taken out of these rooms unless first decontaminated with a minimum of 20 minutes exposure to ultraviolet light per surface area Amplified DNA samples are autoclaved in these rooms prior to disposal Page 13 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 The following special precautions apply while working in the PCR amplification and typing room Clean dedicated lab coats are worn in the PCR amplification and typing room and they are disposed of in this room Gloves are removed when leaving this work area to avoid transfer of amplified DNA into other work areas Gloves changed whenever they have become contaminated with amplified DNA in order to reduce the unnecessary dispersal of DNA aroun
266. second reading for each locus must match 8 Mark the profile for transfer to LDIS and SDIS by clicking on LDIS and SDIS in the Reading Information table A green check mark must be in the box for each locus indicating that the alleles are marked for transfer 9 Click on the Transfer to LDIS button The profiles are now in LDIS 10 Click on the Clear button to clear the entry screen for additional profiles 11 Exit all screens when profile entry is complete Upon completion of a forensic case the analyst will determine which sample s and which alleles in each sample will be entered into LDAS Profiles must be technically reviewed prior to transfer from LDAS to LDIS Each analyst will be responsible for his her case profile entry into the appropriate database in CODIS Binders containing copies of DNA case reports will be maintained at each laboratory Once the sample has been entered into CODIS the copy of the report and the STR allele worksheet will be filed into the DNA case binders Page 168 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 NOTE Copies of the case reports from all cases including those not entered into CODIS must be filed in the DNA case binders G When appropriate the case report will reflect that a search was performed and that the sample will be searched routinely H Only DNA Analysts who have passed the FBI background check will enter profile
267. sened B After removal of the coverslip remove the hair and rinse it thoroughly with xylene C Continue processing at Step B of the procedure for Unmounted Hair Specimens BONE Bone specimens should be store at 20 C until processed A Cut the bone specimen to a size of approximately 2 cm x 5 cm Best results are obtained if the bone is pulverized in a sterile fashion B Transfer the bone specimen to a 2 2 ml or 1 5 ml microcentrifuge tube Page 49 of 255 CBI DNA Analysis SOP Vers 2 1 Od m PX FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 To the sample add 300 yl organic stain extraction buffer 12 ul 1M and 4 ul 10 mg ml Proteinase K solution Vortex for 1 second and spin in a microcentrifuge for 2 seconds to force the sample into the extraction fluid Incubate the tube at 56 C overnight Spin in a microcentrifuge for 2 seconds to force condensation into the bottom of the tube In a fume hood add 300 ul phenol chloroform isoamyl alcohol to the extract Vortex low speed the mixture briefly to attain a milky emulsion Spin the tube in a microcentrifuge for 3 minutes Note If necessary additional organic extractions may be performed prior to the purification steps To a Microcon concentrator add 100 ul Transfer the aqueous phase from the tube in Step F to the concentrator Avoid pipetting organic solvent from the tube into the concentrator Place a cap on the concen
268. sing a 1000 ul pipettor add ultrapure water to the wells Place the plate into the gel cassette gno OO Mount the cassette onto the 377 DNA Sequencer Run the Plate Check module If all 4 colors show a flat line the plate is sufficiently clean If spikes appear remove the plate clean the read region again with ultrapure water and Kimwipes then repeat the Plate Check module Pre Run the gel 1 Mount the upper buffer chamber onto the glass plates and add 1X TBE to the fill line 2 Rinse the wells with 1X TBE A small amount of Formamide Loading Solution may be placed in the wells to make them visible Place the cover on the buffer chamber 3 Fill the lower buffer reservoir with 1X TBE Attach the front heat transfer plate and plug in the ground wire Plug in the upper buffer chamber electrode close the door and start the Pre Run module 6 Pre Run the gel Once the temperature reaches 50 C samples may be loaded Page 137 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 Load the gel 1 Click on the Pause button Once the instrument pauses open the door unplug the upper buffer chamber and remove the lid Rinse the wells with 1X TBE 2 Load the samples using flat gel loading pipette tips The amount loaded per well is dependent upon the comb used and is determined empirically Load all odd wells in sequence Click on the resume button and Pre Ru
269. sis Locus Clinical Chemistry 36 1980 1614 9 Page 90 of 255 CBI DNA Analysis SOP Vers 2 1 24 25 26 27 28 29 30 31 32 FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 Yang F Brune J L Naylor S L Cupples R L Naberhaus K H and Bowman Human Group Specific Component Gc is a Member of the Albumin Family Proceedings of the National Academy of Sciences USA 82 1985 7994 8 Reynolds R L and Sensabaugh G F Use of the Polymerase Chain Reaction for Typing GC Variants Advances in Forensic Haemogenetics 3 Ed Polesky and Mayr W R Heidelberg Springer Verlag Berlin 1990 158 61 Erlich and Bugawan T L HLA Class Gene Polymorphism DNA Typing Evolution and Relationship To Disease Susceptibility PCR Technology Principles and Applications for DNA Amplification Ed Erlich H A New York NY Stockton Press Inc 1989 193 208 Fildes N Reynolds R and Erlich H A n preparation Roche Molecular Systems Inc Higuchi R and Kwok S Avoiding False Positives with PCR Nature 339 1989 237 8 Watson R The Formation of Primer Artifacts in Polymerase Chain Reactions Amplifications A Forum for PCR Users Perkin Elmer Newsletter 2 1989 5 6 Crouse C A Vincek V and Caraballo B K Analysis and Interpretation of the HLA DQa 1 1 Weak signal Observed During the PCR based Typing Method Journ
270. so possible in the case of high target DNA concentration to see n 4 addition and n 8 stutter These stutter peaks may be due to slippage during amplification and sequence microvariants can affect the amount of stutter E g a lower amount of stutter is produced from alleles with increased sequence variation between repeats The following table values should be used as guidelines for the expected levels of stutter measured as a of n Stutter for Profiler Plus Loci Locus 0351358 0851179 021511 018551 055818 0135317 075820 Stutter 12 12 12 10 11 14 10 9 9 Stutter for Cofiler Loci Locus D381358 0165539 1 CSF1PO D7S820 Stutter 12 10 5 6 9 9 Page 112 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 For purposes of mixture interpretation peaks in the n 4 position that exceed the expected percent stutter at a particular locus may be designated as a true allele Stutter peaks may be elevated above established thresholds by the following A Off scale raw data RFU greater than 8100 If the stutter peak is greater than the maximum allowed and the primary peak is above 3000 RFU and or has been labeled off scale the analyst should interpret the results with caution The sample may be diluted and re injected to resolve the issue B
271. ssion spectra The resulting data will be graphically displayed as colored peaks noted by height in relative fluorescent units RFUs and time scan number This display is called an electropherogram The reference allelic ladders for each of the STR loci and reference fragments for amelogenin are also subjected to capillary electrophoresis These allelic ladders contain the more common alleles in the general population for each locus Using the ladders the alleles present in known and questioned DNA specimens may be determined The following table lists the Profiler Plus and Cofiler loci the size ranges of alleles within a particular locus the alleles present in the ladder the color of the labeled primer and the DNA profile of the Positive control 9947A Note that some of the same loci are amplified in both kits Additional information procedures and protocols may be relied on from the Federal Bureau of Investigation Short Tandem Repeat Analysis Protocol July 28 2000 the ABI PRISM 310 Genetic Analyzer User s Manual 1998 ABI PRISM 310 Genetic Analyzer GeneScan Reference Guide 1997 GeneAmp PCR SYSTEM 9700 User s Manual Set 1997 Perkin Elmer DNA Thermal Cycler 480 Quick Reference Guide 1994 and the AmpF STR Profiler Plus and AmpF STR Cofiler User s Manual 1998 Page 94 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE
272. sult in some level of inclusion This is typically reported as the probability based upon genotypic frequencies of observing a random match among unrelated individuals from the population If however the most common frequency for the racial groups determined is 1 000 times greater than the population of the United States then the following statement may be made a reasonable degree of scientific certainty is the source of the DNA item Inconclusive Due to limited amount of information present sometimes only a few loci examined an inclusion statement cannot be issued regarding the comparison however in some instances the results may be defined in terms of the ability or inability to exclude Note In the case of mixtures the results may be described in terms of a known sample being excluded or included as a potential major minor contributor to the genetic material detected in a questioned stain Page 122 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 POPSTATS The CBI may use the POPSTATS software program to calculate population statistics POPSTATS enables one to determine the statistical probability of selecting a person at random from a given population and matching the information contained in a given target profile Alternatively POPSTATS may be used to perform mixture calculations Probability of Exclusion The POPSTATS program has been dis
273. sulting data is graphically displayed as colored peaks noted by height in relative fluorescent units RFUs and time scan number This display is called an electropherogram The reference allelic ladders for each of the STR loci and reference fragments for amelogenin also undergo electrophoresis These allelic ladders contain the more common alleles in the general population for each locus Using the ladders the alleles present in DNA samples may be determined The following tables Table 5 and Table 6 list the Profiler Plus Cofiler and Identifiler loci the size ranges of alleles within a particular locus the alleles present in the ladder the color of the labeled primer and the DNA profile of the positive control 9947A Note that some of the same loci are amplified in both the Profiler Plus and Cofiler Additional information procedures and protocols may be relied on from the AB PRISM 377 DNA Sequencer User Manual 1998 373 and ABI PRISM 377 DNA Sequencers GeneScan Reference Guide 1997 the PRISM 3100 Genetic Analyzer User Manual 2001 GeneAmp PCR System 9700 User s Manual Set 1997 the 5 Profiler Plus User s Manual 1998 the AmpF STR Cofiler User s Manual 1998 and the AmpF STRO Identifiler User s Manual 2001 Page 124 of 255 Table of Contents CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUN
274. t recommended because some inks may affect the quality of the typing results Place one DNA probe strip in each clean well of the AmpliType DNA Typing Tray Strips should all be in the same orientation NOTE AmpliType PM AmpliType HLA DQA1 DNA Probe Strips can be used to type PCR products from the same PM DQA1 amplification reaction at the same time but the DNA probe strips must be placed in separate wells of the tray E Prepare the GeneAmp PCR Instrument System to denature the amplified DNA by setting the temperature parameter to 95 C see Instrument Manual Start the program Place the tubes the GeneAmp PCR Instrument System after it reaches 95 C Press the tubes down tightly in the block Denature the amplified DNA by incubation at 95 C for 3 to 10 minutes Keep each tube at 95 C until use G As shown in Figure 1 tilt the DNA Typing Tray towards the labeled end of the strips The DNA Typing Tray Lid turned upside down may be used as the solid sup port Add 3 ml pre warmed Hybridization Solution to each well at the labeled end of each strip Do not wet the remainder of the strip Page 73 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 NOTE Hybridization solution solids must be completely dissolved before adding to the tray Pipette or aspirate Labeled end of strips DNA Typing Tray Lid Figure 1 Handling Solutions in DNA Typi
275. ted the run folders created can be transferred to the analyst s workstation for analysis and review If the 3100 instrument is not to be used for longer than one week the syringes and the capillary array can be removed for storage See the ABI PRISM 3100 Genetic Analyzer Users Manual for additional storage options Removing and cleaning the syringes Refer to Chapter 8 in the ABI PRISM 3100 Genetic Analyzer Users Manual for additional information regarding cleaning and maintenance 1 If the syringe drives are not in the home positions under Instrument from the menu bar open the Manual Control windows and home both the reserve and capillary fill syringes 2 Unscrew the syringes to remove them from the pump block Expel any unused polymer into a waste container and dispose of according to the MSDS 4 Rinse the syringes with warm tap water by drawing water into the syringe and then expelling it several times Repeat with several rinses of filter purified water 5 If desired the syringes may be dried using compressed air This is not required 6 Store the syringes with the plunger inside Cleaning the Pump Block 1 Within the Manual Control window home both syringes then remove them from the pump block Under Manual Control select Pin Valve Open to open the pin valve on the lower polymer block 2 Remove the previously installed capillary if the array is to be stored off the instrument NOTE If
276. ter the analysis is complete save the project K Transfer a copy of the spectral calibration spatial calibration run notes plate record analysis parameters and size standards to the run folder GENOTYPER ANALYSIS Genotyper software is used to automatically convert the allele sizes from GeneScan Analysis software into allele designations and to build tables containing the Genotyper information Genotypes are assigned by comparing the sizes obtained for the unknown sample alleles with the sizes obtained for the alleles in the allelic ladder The AmpF STR Profiler Plus Cofiler Identifiler NT and Identifiler CODIS NT template files contain the macros that perform the following steps automatically Page 149 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 A Find the sample file containing the ladder The Sample Info column of the GeneScan sample sheet must contain a unique sample description in order for Genotyper software to build a table Also the lanes that contain the allelic ladders must contain the word ladder in the Sample Info column of the sample sheet The Genotyper template identifies the allelic ladder by searching for the word ladder in Sample Info B Create allele size categories that are centered on the sizes obtained for the allelic ladder peaks C Assign the appropriate allele label to sample alleles that fall within the allele size categories D
277. the District Attorney or when there is no named defendant If there is any doubt as to the status of a defendant on any given case the analyst should contact the investigator assigned to the case for specifics Once the District Attorney contacts CBI and informs the analyst in writing that defense has been notified of the situation arrangements shall be made by the analyst and the defense representative to observe only the extraction of the DNA from the minimal evidence The defense representative will observe no further DNA analyses If desired the defense shall be given one half of the extracted DNA to afford them the opportunity to repeat any analyses performed at CBI Under no circumstances shall the defendant s or prosecution s representatives be allowed to be present while DNA tests which can be repeated are being performed This also includes all personnel regardless of whether they are involved in the case Any court orders or attempts by the defense to have their experts present during DNA analysis typing shall be brought to the immediate attention of the Agent in Charge AIC so that the motion may be addressed in a suitable manner Under no circumstances shall the defendant s representatives or any outside experts be allowed to use any equipment or instrumentation owned by CBI Page 16 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 QUALITY CONTROL MEASURES
278. the concentrator from the filtrate cup and carefully invert the concentrator onto a labeled retentate cup Discard the filtrate cup Note Final recovery volumes following purification generally range from 20 ul to 200 ul for evidentiary samples The final recovery volumes for the extraction reagent blanks for evidentiary samples are generally between 20 ul and 200 ul Page 51 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 Spin the assembly in a microcentrifuge at 2500 x g for 5 minutes Discard the concentrator Cap the retentate cup Estimate the quantity of DNA in the sample by slot blot hybridization After quantification the sample can be amplified Store samples at 4 C or frozen Prior to the use of samples after storage they should be vortexed briefly and spun in microcentrifuge for 5 seconds Page 52 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 This Page Intentionally Left Blank Page 53 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 BLOOD EXTRACTION USING FTA CARDS FTA cards are chemically treated to tightly bind DNA Blood from database samples is spotted onto the cards Punches from the cards are subjected to a series of washes that remove cellular components The DNA remains bound to the cards and never enters solution s
279. tion add 10 ml 1M Tris pH 8 0 0 05 20 ml 0 5 M EDTA and 100 20 SDS Titrate to pH 8 0 with Bring to a final volume of 1L with ultrapure water Autoclave Store at room temperature Date 1M Tris pH 8 0 0 5M EDTA 20 SDS Prepared lot pH Preparer Verified date Amount NaCl Lot lot lot ORGANIC STAIN EXTRACTION BUFFER Page 240 of 255 Appendix AG DNA Working Solutions Log Organic Stain Extraction Buffer CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 APPENDIX AH DNA WORKING SOLUTIONS Loc PM DQA1 WASH SOLUTION MODEL 2 5X SSPE 0 1 wiv SDS 2L Solution Preparation Add 250 ml 20X SSPE and 10 ml 20 w v SDS to 1740 ml ultrapure water and mix thoroughly Note Wash solution solids must be in solution before use warming e g 50 C incubator may be required to dissolve solids completely Preparation in a clear glass container is recommended to facilitate visual inspection for solids during warming 20X SSPE 20 SDS Date Prepared Amount lot lot Preparer PMDQAwash Page 241 of 255 Appendix AH DNA Working Solutions Log PM DQA1 Wash Solution CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 APPENDIX Al DNA WORKING SOLUTIONS LOG PROTEINASE 10 MG ML MODEL 10 ML Solution P
280. toclave and store at room temperature EE 1M TRIS HCI pH 7 5 1L 1 Dissolve 121 1 g Tris base in 800 ml ultrapure water Adjust to pH 7 5 room temperature by adding concentrated approximately 65 NOTE Electrodes do not accurately measure the pH of Tris buffer be sure to use suitable electrodes for the Tris buffer pH adjustment 2 Adjust the final volume to 1 liter with ultrapure water Sterilize by autoclaving Store at room temperature FF 1M TRIS HCI pH 8 0 1L 18 Dissolve 121 1 g Tris base 800 ml ultrapure water Adjust to pH 8 0 at room temperature by adding concentrated approximately 45 NOTE Electrodes do not accurately measure the pH of Tris buffer be sure to use suitable electrodes for the Tris buffer pH adjustment 2 Adjust the final volume to 1 liter with ultrapure water Autoclave Store at room temperature Page 31 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 This Page Intentionally Left Blank Page 32 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 INORGANIC EXTRACTION CHELEX All extraction steps must be performed in the DNA extraction PCR setup laboratory using hoods reagents and pipettors dedicated to this area Chelex is a chelating resin that has a high affinity for polyvalent ions The Chelex resin is composed of styrene divin
281. trast D Follow film exposure and development instructions After photography the DNA probe strips may be air dried on any hard absorbent surface Protect from light and oxidizing agents e g acid treated paper bleach and nitric acid The dot intensities may fade upon drying DISPOSAL OR REUSE OF TYPING TRAYS A The AmpliType DNA Typing Trays are disposable or may be reused Used trays and lids should be washed according to the following procedure NOTE Do NOT use detergent 1 To each well of the used AmpliType DNA Typing Tray add approximately 5 to 10 of 95 ethanol or 70 isopropanol 2 Cover the tray with the lid and carefully agitate for 15 to 30 seconds to dissolve any residual Chromogen TMB 3 Remove the lid and pour off the ethanol or isopropanol from each well Visually inspect each well for a faint blue color that will indicate the presence of Chromogen TMB If necessary repeat Steps A1 and A2 to remove any residual Chromogen TMB 4 Rinse each well in the tray and the tray lid with either glass distilled or deionized ultrafiltered water Repeat 5 Dry trays before reuse INTERPRETATION OF RESULTS Results are interpreted by observing the pattern and relative intensities of blue dots on the wet AmpliType PM and AmpliType HLA DQA1 DNA Probe Strips to determine which alleles are present in the DNA sample Page 78 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA
282. trator and spin in a microcentrifuge at 2500 x g for 10 minutes Add 100 ul to the concentrator Replace the spin cap and spin the assembly in a microcentrifuge at 2500 x g for 10 minutes Remove the and add a measured volume of that is between 20 ul and 200 ul to the concentrator Remove the concentrator from the filtrate cup and carefully invert the concentrator onto a labeled retentate cup Discard the filtrate cup Note Final recovery volumes following purification generally range from 20 ul to 200 ul for evidentiary samples The final recovery volumes for the extraction reagent blanks for evidentiary samples are generally between 20 ul and 200 ul Spin the assembly in a microcentrifuge at 2500 x g for 5 minutes Discard the concentrator Cap the retentate cup Estimate the quantity of DNA in the sample by slot blot hybridization After quantification the sample can be amplified Store samples at 4 C or frozen Prior to the use of samples after storage they should be vortexed briefly and spun in a microcentrifuge for 5 seconds Page 50 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 TEETH In general for DNA analysis large teeth with no restorations should be chosen over smaller restored teeth Thus a non restored molar tooth is the tooth of choice for DNA recovery A Clean the outer surface of the tooth with alcohol B Split or crush
283. tributed to forensic laboratories as part of the CODIS software Calculating genotype frequencies for casework DNA profiles A Currently the frequency data from the FBI databases is used B The unbiased expected frequency is determined directly from the allele frequencies of the population data using the CODIS POPSTATS 5 2 software program 1 The NRC Il 1996 4 2 Option featuring the theta value for homozygote frequencies is selected A theta value of 0 01 is used for all populations 2 Refer to the POPSTATS 5 1 Calculation Specification Manual for all statistical formulas 3 A 0 01 default value is used for alleles that have not been detected within the populations or have been detected at a frequency below 0 01 C A POPSTATS report will be provided for all cases in which statistical calculations have been conducted This report will give frequency data for Caucasian African American and southwestern Hispanic populations This report will be kept in the case file and will be peer reviewed NOTE Please refer to sections Interpretation of the Specimens on page 13 and Application of Population Frequency Data to Profiler Plus and Cofiler Typing Results on page 13 for information on statistical methods and casework interpretation and statistical references Page 123 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 PRINCIPLES OF STR ANALYSIS DATABASE AmpF STR
284. ual is the biological sibling or some other distant relative of someone that is the source of a stain tissue or biological substance These cases shall be referred to an external agency or laboratory that participates in these evaluations on a regular basis The Colorado Bureau of Investigation will indicate no preference to which agency or laboratory is used Performing the genetic analysis using current CBI standard procedures and reporting only the genetic profile of each sample tested can provide the assistance to the requesting agency The requesting agency can utilize this information in soliciting statistical inference on the relationships between individuals and samples submitted ELECTRONIC FILE NAMING CONVENTION FOR THE 310 STR ANALYSIS The case number forms the basis for naming electronic files In the example below the case number is D50 5010 Date is listed as month day year for Mac and month day year for IBM ai analyst s initials lower case PP Profiler Plus CO Cofiler Case D50 5010 Sample Number 1D505010 item 1 from case D50 5010 Sample Number Semen IE SD505010 Sample Sheet SS dateaiPPand orCO Run Folder Run Folder dateaiPP and or CO GeneScan Project GS Project dateaiPPand or CO Matrix file Matrix dateaiinstrument Size Standard GS500dateaiPP or CO Genotyper document edited GT dateaiPP or CO Genotyper document case GT dateaiPP or COD50 5010 Mac date electropherograms Genotyper document
285. ube If needed dilute a portion of the sample DNA with autoclaved DI so that only 2 ng to 10 ng of DNA will be added to the PCR reaction a volume of 18 ul 2 POSITIVE CONTROL TUBE vortex the Control DNA 1 and spin the tube briefly in a microcentrifuge before use to remove any liquid from the cap Add 18 yl of the 100 ng ml 1 8 ng Control DNA 1 to the labeled positive control tube 3 NEGATIVE CONTROL TUBE add 18 yl of autoclaved DI to the labeled negative control tube As soon as all samples have been added place the tubes into the GeneAmp PCR Instrument System Push the tubes down completely into the block The position of each tube in the block should be recorded Page 71 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 J Start the 32 cycle amplification file Verify the cycling parameters by monitoring the first cycle Check the tubes after the first cycle to insure they are all still seated tightly in the block PREPARATION OF PCR PRODUCT FOR DETECTION After the PCR amplification process remove the tubes from the GeneAmp PCR Instrument System Prior to DNA hybridization Section DNA Hybridization open the tubes one at a time and add 5 ul 200mM disodium EDTA Use a new pipette tip for each addition Carefully insert the pipette tip through the mineral oil layer Discard the pipette tip and recap the tube before proceeding to the next tube NOTE
286. ult of spectral overlap between the dyes which are normally corrected for by the matrix If a pull up peak is above the minimum peak height detection threshold it will be sized at approximately the same size usually within 0 1 bp but may be more as the true peak Pull up can occur as a result of the following e Application of a sub optimal matrix e Amplification using excess input DNA which may lead to off scale peaks SPIKES AND OTHER ANOMALIES Inconsistencies in the gel or polymer as well as electrical impulses can cause artifactual peaks Most artifactual peaks can be shown to be false by rerunning the sample The peaks fail to show in the same bp size location Spikes can occur in one two three or all four or five colors Artifactual peaks occurring in all four or five colors are the easiest to diagnose as they will be the same size and of similar height Artifactual peaks occurring in less than four or five colors should be interpreted with caution especially single color spikes and the analyst may choose to rerun the sample to insure a clear interpretation Generally spikes are thin peaks with variable peak heights often having the same scan number OFF SCALE DATA Multicomponent analysis of off scale data may result in raised baselines excessive pull up of one or more colors under the off scale peaks or unnaturally high stutter peak ratios Analysts may choose to interpret samples with off scale data approx 5000
287. ust not come in direct contact with each other or with unprepared cards The blood spotted stain card is left in the covered hood to dry at least one hour Page 178 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 Upon completion of preparing the stain card the offender s blood tube and any remaining blood are disposed of following the procedures established by the biohazardous waste disposal contractor for the site Once the spotted stain card is thoroughly dry the card is packaged in a plastic sleeve heat sealed with the preparer s initials amp date and stored appropriately The FTA cards are stored in a locked cabinet at room temperature The S amp 51 cards are stored in a locked 70 C freezer C INTERLAB TRANSFER OF SAMPLES 1 DBS samples may be transferred between CBI laboratories to facilitate the effective use of personnel resources and materials DBS samples are transferred by common carrier e g UPS or Federal Express or by an agent of the Bureau traveling to a particular site Whole blood must be kept refrigerated Prepared stain cards made paper may be kept at room temperature Each DBS sample must be transferred with the corresponding working copy from the Database Submission Form There is no internal chain of custody and no signature required for interlab transfers or upon completion of analysis The laboratory assigned
288. verification of matches involving profiles entered into their system The Local CODIS Administrator is also responsible for SDIS uploading CODIS Administrator State or SDIS Responsible for overseeing the CODIS system Duties may include organization of CODIS materials installing software routine tape backup system QA routine local searches statewide autosearches and training of Local Administrators The State CODIS Administrator is also responsible for NDIS uploading verification of matches and certifying that all CODIS laboratories in the state are in compliance with NDIS requirements CODIS Import Import is a Windows program that copies DNA data from a Common Message Format CMF data file and places the data into LDAS CODIS core loci The following STR loci constitute the 13 core loci that are required for a complete PCR profile CSF1PO FGA 1 vWA 0351358 055818 075820 0851179 0135317 0165539 018551 and 021511 CODIS User CODIS user is anyone who has access to CODIS and has passed the FBI background check The State Administrator adds users to NDIS Forensic Unknown sample A biological sample that is found at the scene of a crime that does not appear to have originated from the victim or any elimination individuals GDIS Generalized DNA Index System The software and database used for LDIS SDIS and NDIS Hit A confirmed match between two or more DNA profiles discov
289. when completed because this number will be used to import the sample into the CODIS database NOTE Refer to the Naming Convention for STR Analysis of Database Samples section for the sample name format Insure that the information contained under the Sample Name column is copied onto the Sample Info column 2 Fill in the plate record according to the well number where you want to place the samples Make sure each set of 16 samples has at least one ladder Be sure that all colors that correspond to the fluorescent labels in the PCR kit used in the amplification process are present in the Color Number column Page 141 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 Color Comment may be left blank but filling in this field may prevent column shifting during the plate import process Note Under the Sample Info column insure that each sample has a unique sample number also insure that each well which contains an allelic ladder has the word ladder in the Sample Info column this is necessary for automated allele calling with Genotyper v 2 0 or higher 3 Select a Dye Set that corresponds to the PCR amplification kit used to amplify the sample Dye Set G5 is used for Identifiler and Dye Set F is used for Cofiler and Profiler Plus kits Select 3100 Project 1 under the BioLIMS project column Select a Run Module Genescan Run Modules are determined
290. wo DNA Calibrators Add 5 ul of each solution to the corresponding labeled tube containing 150 ul Spotting Solution NOTE Sample DNA should be MgCl2 free See Troubleshooting Section 4 Add 1 to 5 ul of each test sample DNA to the remaining tubes containing 150 yl Spotting Solution b While wearing clean gloves cut a piece of Biodyne B membrane to 11 0 cm x 7 9 cm Cut a small notch in the upper right corner of the membrane to mark orientation Place the membrane in the Hybridization Tray Part No N808 0136 containing 50 ml Pre Wetting Solution Incubate at room temperature for 1 to 30 minutes NOTE The following protocol is for use with GIBCO BRL the Convertible slot blot apparatus Refer to GIBCO BRL instructions for additional details Page 58 of 255 CBI DNA Analysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 6 Using forceps remove the membrane from the Pre Wetting Solution Place the membrane on the gasket of the slot blot apparatus then place the top plate of the slot blot apparatus on top of the membrane Turn on the vacuum source i e turn on house vacuum line or vacuum pump Turn off the sample vacuum and turn on the clamp vacuum on the slot blot apparatus Push down on the top plate to insure the formation of a tight seal Pour off the Pre Wetting Solution and rinse the Hybridization Tray thoroughly with DI H20 7 Use new pipette tip for each sample Pipet eac
291. x C Resources CBI DNA SOP Vers 2 1 06 13 02 4 43 PM CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 Mannucci A Sullivan K M lvanov P L Gill P Forensic Applications of a Rapid and Quantitative DNA Sex Test by Amplification of the X Y Homologous Gene Emelogenin nt J Leg Med 106 1994 190 3 Nakahori Y Hamano K lwaya M Nakagome Y Sex Identification by Ploymerase Chain Reaction Using X Y Homologous Primers Amer J Med Genet 39 1991 472 3 Salido E et al The Human Enamel Protein Gene Amelogenin is Expressed from Both X and Y Chromosomes Amer J Hum Genet 50 1992 303 16 Sullivan K Mannucci A Kimpton C P Gill P A Rapid and Quantitative DNA Sex Test Fluorescence Based PCR Analysis of X Y Homologous Gene Amelogenin Biotechniques 15 1993 636 42 EXTRACTION Comey C T Koons B W Presley K W Smerick J B Sobieralski C A Stanley D M and Baechtel F S DNA Extraction Strategies for Amplified Fragment Length Polymorphism Analysis J Forens Sci 39 1994 1254 69 DNA QUANTIFICATION Budowle B Baechtel F S Comey C T Giusti A M and Klevan L Simple Protocols for Typing Forensic Biological Evidence Chemiluminescent Detection for Human DNA Quantification and RFLP Analyses and Manual Typing PCR Amplified Polymorphisms Electrophoresis 16 9 1995 1559 67 Walsh P S Varlaro J and Reynolds R A Rapid Chem
292. ylbenzene copolymers containing paired iminodiacetate ions which act as chelating groups It has been postulated that the presence of Chelex during boiling prevents the degradation of DNA by chelating metal ions that may catalyze the breakdown of DNA subjected to high temperatures in low ionic strength solutions The basic Chelex procedure consists of boiling the sample in a 5 Chelex solution and then adding a fraction of the supernatant directly to the PCR Mix This Chelex procedure results in denatured sample DNA BLOOD PROCEDURE A Add about a 3 mm square of bloodstain or ul whole blood to a sterile 1 5 ml microcentrifuge tube Add 1 ml of TE Buffer Incubate at room temperature for 15 to 30 minutes Mix occasionally by inversion or gentle vortexing C Spin in a microcentrifuge for 2 to 3 minutes at 10 000 to 15 000 x g D Carefully remove supernatant all but 20 to 30 ul and discard If the sample is a bloodstain leave the fabric substrate in the tube with the pellet Add 5 Chelex to a final volume of 200 ul F Incubate at 56 C for 15 to 30 minutes or overnight Overnight is optimal Vortex at high speed for 5 to 10 seconds Spin in a microcentrifuge for 5 to 10 seconds at 10 000 to 15 000 x g H Incubate in a boiling water bath for 8 minutes l Vortex at high speed for 5 10 seconds Spin in a microcentrifuge for 2 to 3 minutes at 10 000 to 15 000 x g The sample is now ready for the PCR Qu
293. ysis SOP Vers 2 1 CBI FORENSIC LABORATORY DNA ANALYSIS SOP VERSION 2 1 JUNE 2002 T Check the antifreeze bottle on the right side of the instrument It should be at least 2 3 full If necessary replenish with 5 antifreeze diluted in ultrapure water STR TYPING BY CAPILLARY GEL ELECTROPHORESIS USING THE ABI PRISM 3100 GENETIC ANALYZER A B Start the computer Make sure the Orbixweb software has launched before proceeding Turn on the ABI PRISM 3100 Genetic Analyzer Wait until the green light on the 3100 is on before proceeding See Chapter 3 in the ABI PRISM 3100 Genetic Analyzer User s Manual under Starting the 3100 System for more information Check the hard drive to make sure there is enough room on the computer for sample data by running the Diskspace Utility D AppliedBio 3100 Bin If there is not enough computer memory space you may have to run Cleanup DataBase D appliedBio 3100 Bin Utility Refer to the procedure in the ABI PRISM 3100 Genetic Analyzer User s Manual Chapter 7 Reminder this utility will delete all run data and plate records from the database However it will not affect spatial and spectral information Launch the 3100 collection software Remove the POP 4 from the refrigerator to warm to room temperature If crystallized urea is present when the bottle is removed warming to room temperature and gentle mixing rotate slowly and carefully by hand to avoid introducing

Colordao Bureau of Investigations SOP for DNA (June, 2002)

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