Home

AmpFlSTR® Identifiler™ PCR Amplification Kit

1. Step Action 7 From the Sample Sheet drop down menu in the GeneScan Injection List import the appropriate sample sheet Note To access five dye modules you must first import a five dye sample sheet into the injection list En Injection List 9 18 00 10 46 AM Sample Sheet Lane a pb pon Pause Bl Cancel linj Tube amp Sample Name 1 8 After setting the appropriate injection parameters save the injection list 9 To start the sequence of injections click the Run option in the Injection List window Protocol for 310 Genetic Analyzer with Mac OS 3 17 Making a Matrix File Matrix Standards The matrix standards are supplied in the Matrix Standard Set DS 33 6 FAM VIC NED PET and LIZ for use with the 310 377 system P N 4318159 Making a Matrix To make the matrix file File on the ABI Prism 310 SteP Action 1 Combine 1 uL of each matrix standard with 25 uL of Hi Di Formamide P N 4311320 Prepare one tube for each matrix standard sample CHEMICAL HAZARD Formamide is harmful if absorbed through the skin and may cause irritation to the eyes skin and respiratory tract It may cause damage to the central nervous system and the male and female reproductive systems and is a possible birth defect hazard Please read the MSDS and follow the handling instructions Wear appropriate protective eye wear cl
2. 2200 NED dye 2400 1600 s00 0 1 3200 2400 1600 PET dye 800 d AM 1 Tit Figure 3 4 This figure exhibits the raw data of each matrix standard analyzed on the ABI Prism 310 Genetic Analyzer 9 When the injections are done follow these steps in the GeneScan Analysis Software a Under the File menu select New b Click the Matrix icon Select five dyes from the number of dyes pop up window In the window that appears indicate the sample files that correspond to each matrix standard dye color c Select starting scan numbers for each sample to exclude the primer peak as represented in Figure 3 4 d Select the number of points such that at least these five peaks are contained in the scanned region this is approximately 2500 scan data points Avoid spikes or artifacts if possible when selecting the range e Click OK The computer makes the matrix and the matrix file table appears 8200 2400 1600 800 0 H Protocol for 310 Genetic Analyzer with Mac OS 3 19 To make the matrix file continued Step Action 10 Save the matrix file in the ABI folder within the System folder To verify the accuracy of the matrix file Step Action 1 Apply the new matrix file to the Matrix Standard Sample Files as follows a In the Analysis Control window highlight the Sample File co
3. Protocol for 310 Genetic Analyzer with Mac OS 3 5 To clean the syringe Step Action 2 Remove the ferrule from the syringe a Soak the ferrule in warm not boiling water for as long as it takes to remove crystals if any in the ferrule b Rinse the ferrule with deionized water Clean the glass barrel with warm water Dissolve any crystals Rinse the glass barrel with distilled deionized water IMPORTANT Remove all residual water from the syringe by blowing compressed air through it Inspect the O ring in the stainless steel hub of the syringe for damage and replace it if necessary IMPORTANT Make sure the O ring does not block the hole in the stainless steel hub Inspect the O ring on the ferrule and replace it if necessary Place the ferrule back onto the syringe IMPORTANT The Teflon tip of the plunger must be damp when inserting it into the barrel place a drop of distilled deionized water on the Teflon or the Teflon tip could be damaged Note For syringe storage the plunger should remain in the syringe barrel 3 6 Protocol for 310 Genetic Analyzer with Mac OS Loading the Syringe To load the syringe Step Action 1 Prime the syringe with approximately 0 1 mL of POP 4 polymer 2 Fill the 1 0 mL syringe manually with a maximum of 0 8 mL of POP 4 polymer Note The polymer should not stay in the syringe longer than 3 days Do not return
4. 8d 15 19 Amelogenin X p22 1 22 3 Y p11 2 X Y D58818 5q21 31 7 8 9 10 11 12 13 14 15 16 FGA 4028 17 18 19 20 21 22 23 24 25 26 26 2 27 28 29 30 30 2 31 2 32 2 33 2 42 2 43 2 44 2 45 2 46 2 47 2 48 2 50 2 51 2 PET 23 24 a For CODIS purposes profile reported as 13 13 b For CODIS purposes profile reported as 30 30 c For CODIS purposes profile reported as 11 11 d For CODIS purposes profile reported as 8 8 e For CODIS purposes profile reported as 11 11 Introduction 1 5 About This User s This user s manual provides users of the AmpFZSTR Identifiler PCR Manual Amplification Kit with protocols and data obtained by Applied 1 6 Introduction Biosystems Applied Biosystems recommends that users conduct similar experiments to evaluate in their labs the DNA typing system consisting of AmpFZSTR Identifiler PCR Amplification Kit reagents software and ABI PRISM instruments Furthermore Applied Biosystems suggests that users apply the standards by the community for which this test will be used to further evaluate this DNA typing system This user s manual describes the following Materials and equipment required to use the AmpFZSTR Identifiler kit Howto use the kit to amplify DNA samples How to perform automated detection How to analyze results Materials for the Kit Kit Contents The AmpFZSTR Identifil
5. STR Identifiler kit results from a 1 2 mm FTA bloodstain punch 25 cycle amplification analyzed on the ABI PRISM 310 Genetic Analyzer 2 8 PCR Amplification Protocol for 310 Genetic Analyzer with Mac OS Overview About This AmpFZSTRS Identifiler PCR Amplification Kit products are Chapter electrophoretically separated using a capillary filled with POP 4 Performance Optimized Polymer 4 and detected on the ABI PRISM 310 Genetic Analyzer Protocols for analyzing samples on the ABI PRISM 310 Genetic analyzer are described in this chapter In This Chapter This chapter contains the following topics Topic See Page Overview 3 1 Software Requirements 3 2 Preparing the ABI PRISM 310 Genetic Analyzer 3 3 Setting Up the Run 3 11 Filter Set G5 Module Files 3 14 Five Dye Data Collection 3 15 Making a Matrix File 3 18 Running DNA Samples 3 22 Setting Up Software Parameters 3 26 GeneScan Software Results 3 30 Off Scale Data 3 33 Shutting Down the Instrument 3 34 Dedicated Equipment and Supplies 3 35 Protocol for 310 Genetic Analyzer with Mac OS 3 1 Software Requirements Collection Data Collection Software v2 1 must be installed before AmpFZSTR Software ldentifiler PCR Amplification Kit products can be run in order to properly collect five dye data Additionally before running AmpFZSTR Identifiler PCR products on the instrument a matrix file must be made using the 6 FAM
6. The AmpF STR Identifiler PCR Amplification Kit is covered by U S Patent No 5 364 759 owned by Baylor College of Medicine and is sold under license from Baylor College of Medicine AmpF STR Kits are developed and manufactured by Applied Biosystems Foster City CA USA Certain Applied Biosystems PCR reagents are developed and manufactured by Roche Molecular Systems Inc ABI PRISM and its Design AmpF STR Applied Biosystems BioCAD CytoFluor GeneScan Genotyper MicroAmp POROS and Procise are registered trademarks of Applera Corporation or its subsidiaries in the U S and certain other countries AB Design Applera COfiler 6 FAM FMAT Hi Di Identifiler LIZ Mariner NED PET Pioneer POP 4 Profiler Plus SGM Plus VIC and Voyager are trademarks of Applera Corporation or its subsidiaries in the U S and certain other countries AmpliTaq AmpliTaq Gold GeneAmp QuantiBlot and TaqMan are registered trademarks of Roche Molecular Systems Inc Mac and Macintosh are registered trademarks of Apple Computer Inc All other trademarks are the sole property of their respective owners Contents 1 Introduction uu stain R 1 1 About This Chapter epus E RA REG eek GONE 1 1 In This Chapter out volcs RR BEES Aes ARIE eR 1 1 Product Overview nist scsneseest etre ev be ee ER 1 2 P rpose uer aeree Pede i OO ES UR ER RO e EU RR PERRA 1 2 Five Dye DNA Fragment Analysis eese 1 2 About the
7. BW 10uM GM 46 43 BW 10umM BW 10uM LI 4 43 BW 10uM BW 10uM BE 45 49 Bw 10uM BW 10uM 16003 0i A A ii A a a A a A aa 5B A5 UR sr as BW 12uM BW 12uM BW 12uM BW 12uM BE 56 45 Bw 12uM BW 12uM BE OSR AS Bw 12uM BW 12uM ke r a z m oj A aa i 6B A7 BW 14uM BW 14uM EH 8G 47 BW 14uM BW 14uM UR 6Y A7 BW 14uM EW 14uM GR a7 Bw 14uM BW 14uM 4800 32001 16003 Oi 4 a i a 7B AS BW 16uM BW 16uM DE 76 43 Bw 16uM Bw 16uM DWM 7v 43 Bw 16uM EW 16uM BE 7R AS Bw 16uM BW 16uM of a a a 8B A11 OM vct BW 18uM BW 18uM BW 18uM BW 18uM GM 86 11 Bw 18uM BW 18uM sR A111 BW 18uM BW 18uM 9B B2 BW 20uM BW 20uM OW 3v B2 Bw 20uM BW 20uM GM 36 82 Bw 20uM BW 200M BE 3R 82 Bw 20uM BW 20uM Figure 4 13 DNA amplified with the AmpF STR Identifiler kit in the presence of varying concentrations of hematin 0 10 uM 12 uM 14 uM 16 uM 18 uM and 20 uM analyzed on the ABI PRISM 310 Genetic Analyzer Degraded DNA As the average size of degraded DNA approaches the size of the target sequence the amount of PCR product generated is reduced This is due to the reduced number of intact templates in the size range necessary for amplification Degraded DNA was prepared to examine the potential for differential
8. Budowle B DeFenbaugh D A Keys K M 2000 Genetic variation at nine short tandem repeat loci in Chammorros and Filipinos Legal Medicine 2 1 26 30 Buel E Wang G and Schwartz M 1995 PCR amplification of animal DNA with human X Y amelogenin primers used in gender determination J Forensic Sci 40 641 644 Buel E Schwartz M B and LaFountain M J 1998 Capillary STR analysis Comparison to gel based systems J Forensic Sci 43 1 164 170 Buel E LaFountain M Schwartz M and Walkinshaw M 2001 Evaluation of capillary electrophoresis performance through resolution measurements J Forensic Sci 46 2 341 345 Butler J 2001 Forensic DNA Typing Academic Press San Diego CA Chakraborty R Smouse P E and Neel J V 1988 Population amalgamation and genetic variation observations on artificially agglomerated tribal populations of Central and South America Am J Hum Genet 43 709 725 Chakraborty R Fornage M Guegue R and Boerwinkle E 1991 Population genetics of hypervariable loci analysis of PCR based VNTR polymorphism within a population In Burke T Doif G Jeffreys A J and Wolff R eds DNA Fingerprinting Approaches and Applications Birkhauser Verlag Berlin pp 127 143 Chakraborty R and Stivers D N 1996 Paternity exclusion by DNA markers effects of paternal mutations J Forensic Sci 41 671 677 Chakraborty R Stivers D and Zhong Y 1996
9. C in a glass bowl Take the Wash Solution off the heat source and place the nylon QuantiBlot membrane containing the spotted samples into the solution Rotate on an orbital shaker at room temperature for 20 min Remove the membrane from the Wash Solution IMPORTANT Do not let the QuantiBlot membrane dry out at any time Begin the QuantiBlot kit protocol starting at the hybridization step refer to the QuantiBlot Human DNA Quantitation Kit product insert Performing Is it possible to spot the samples onto the membrane and then perform the hybridization and detection steps at a later time Hybridization and Detection ata Yes Proceed as follows Later Time E 6 DNA Quantitation Step Action 1 Immediately after spotting the samples onto the membrane place the membrane in 100 mL of 5X SSPE without SDS Store at 2 6 C protected from light Resume the protocol beginning with the pre hybridization step Section 4 1 in the QuantiBlot Human DNA Quantitation Kit product insert For best sensitivity resume the protocol within 24 hr References Akane A Matsubara K Nakamura H Takahashi S and Kimura K 1994 Identification of the heme compound copurified with deoxyribonucleic acid DNA from bloodstains a major inhibitor of polymerase chain reaction PCR amplification J Forensic Sci 39 362 372 American Association of Blood Banks 1999 S
10. 1 2 and its associated subsections Whereas this DNA methodology is not novel Standard 8 1 2 and its related subsections have been addressed Holt et al 2001 and Wallin et a 2001 This chapter will discuss many of the experiments performed by Applied Biosystems and examples of results obtained Conditions were chosen which produced maximum PCR product yield and a window in which reproducible performance characteristics were met These experiments while not exhaustive are appropriate for a manufacturer in our opinion Each laboratory using the AmpFZSTR Identifiler PCR Amplification kit should perform appropriate validation studies 4 2 Experiments and Results Developmental Validation 8 1 1 Developmental validation that is conducted shall be appropriately Developmental documented DNA Advisory Board 1998 TOOTH Critical reagent concentrations and reaction conditions e g thermal cycling parameters AmpliTaq Gold DNA polymerase activation cycle number to produce reliable locus specific amplification and appropriate sensitivity have been determined PCR Components The concentration of each component of the AmpFZSTR Identifiler kit was examined The PCR components are Tris HCI pH 8 3 KCl dNTPs primers AmpliTaq Gold DNA Polymerase MgCL bovine serum albumin and sodium azide The concentration for a particular component was established to be in the window that meets the reproducible performance characteristics of specif
11. 24 242 83 0 07 25 246 88 0 06 26 250 96 0 06 26 2 253 00 0 09 27 254 97 0 08 28 259 02 0 10 29 263 12 0 08 30 267 26 0 09 30 2 269 07 0 10 31 2 273 17 0 09 32 2 277 24 0 08 33 2 281 33 0 09 42 2 319 83 0 14 43 2 324 04 0 14 44 2 328 26 0 13 45 2 332 42 0 16 46 2 336 43 0 14 47 2 340 42 0 14 48 2 344 15 0 10 50 2 351 45 0 05 51 2 355 13 0 05 4 16 Experiments and Results Table 4 1 Example of precision results of seven injections of the AmpF STR Identifiler Allelic Ladder continued ABI Prism 310 Genetic Analyzer Allele Mean S D THO1 4 163 29 0 04 5 167 36 0 03 6 171 40 0 05 7 175 40 0 03 8 179 38 0 04 9 183 36 0 05 9 3 186 93 0 02 10 187 29 0 04 11 191 23 0 03 13 3 201 94 0 05 TPOX 6 222 07 0 04 7 226 02 0 06 8 229 91 0 03 9 233 86 0 06 10 237 88 0 07 11 241 83 0 06 12 245 77 0 07 13 249 78 0 08 Experiments and Results 4 17 Table 4 1 Example of precision results of seven injections of the AmpF STR Identifiler Allelic Ladder continued ABI Prism 310 Genetic Analyzer Allele Mean S D vWA 11 154 59 0 08 12 158 87 0 07 13 163 00 0 05 14 167 27 0 05 15 171 15 0 05 16 175 15 0 04 17 179 15 0 04 18 183 08 0 04 19 187 00 0 04 20 190 93 0 05 21 194 80 0 05 22 198 62 0 06 23 202 44 0 05 24 206 69 0 08 4 18 Experiments and Results Extra Peaks in the Electropherogram Causes of Extra To further demonstrate reproducibility 1187 population database DNA Peaks samples hav
12. 25 337 67 0 11 26 341 56 0 09 27 345 24 0 08 Experiments and Results 4 13 Table 4 1 Example of precision results of seven injections of the AmpF STR Identifiler Allelic Ladder continued ABI Prism 310 Genetic Analyzer Allele Mean S D D19S433 9 101 99 0 05 10 105 88 0 05 11 109 78 0 04 12 113 64 0 02 12 2 115 61 0 02 13 117 56 0 03 13 2 119 55 0 02 14 121 46 0 03 14 2 123 47 0 02 15 125 45 0 05 15 2 127 43 0 05 16 129 44 0 05 16 2 131 46 0 05 17 133 42 0 03 17 2 135 44 0 06 4 14 Experiments and Results Table 4 1 Example of precision results of seven injections of the AmpF STR Identifiler Allelic Ladder continued ABI Prism 310 Genetic Analyzer Allele Mean S D D21S11 24 184 86 0 04 24 2 186 82 0 02 25 188 77 0 03 26 192 69 0 05 27 196 56 0 04 28 200 41 0 05 28 2 202 36 0 05 29 204 32 0 03 29 2 206 31 0 02 30 208 29 0 07 30 2 210 24 0 05 31 212 23 0 05 31 2 214 14 0 06 32 216 14 0 04 32 2 218 10 0 04 33 220 14 0 05 33 2 222 07 0 04 34 224 10 0 07 34 2 226 02 0 06 35 228 07 0 06 35 2 230 01 0 07 36 232 04 0 07 37 236 00 0 03 38 239 94 0 08 Experiments and Results 4 15 Table 4 1 Example of precision results of seven injections of the AmpF STR Identifiler Allelic Ladder continued ABI Prism 310 Genetic Analyzer Allele Mean S D FGA 17 214 81 0 07 18 218 80 0 06 19 222 79 0 07 20 226 81 0 06 21 230 76 0 08 22 234 78 0 07 23 238 81 0 05
13. 29 0 17 e 14 12 04 15 76 7 41 6 81 15 30 53 25 36 39 14 40 84 15 2 0 14 a i 16 28 57 22 78 26 72 32 98 17 19 47 18 19 16 03 9 95 18 6 72 16 48 8 97 8 38 19 0 84 1 00 1 03 0 79 20 b 5 0 34 T D5S818 7 0 14 N 6 72 15 71 8 5 46 E 0 69 9 1 68 4 15 5 17 6 02 10 6 72 5 44 5 17 4 19 11 25 49 39 26 39 14 41 10 12 36 41 35 24 29 31 23 30 13 21 57 15 47 12 59 9 42 14 2 38 0 14 0 69 0 26 15 t 0 29 0 18 2 16 9 zi 0 17 g 17 0 14 0 17 4 46 Experiments and Results Table 4 3 AmpFZSTR Identifiler kit allele frequencies continued African American U S Caucasian U S Hispanic Native American Allele n 2 357 n 2 349 n 290 n 191 D7S820 6 i 0 14 0 17 i 7 0 42 1 29 1 72 0 52 8 18 77 16 48 11 72 13 09 9 13 73 17 62 6 21 8 12 10 34 45 27 22 27 41 21 99 11 19 89 18 05 28 79 28 80 12 10 78 14 76 20 17 24 08 13 1 54 3 72 3 45 3 40 14 0 42 0 72 0 34 d 15 D8S1179 8 0 42 2 29 0 34 0 52 9 0 42 1 15 0 34 0 26 10 2 38 9 74 8 45 4 71 11 3 92 6 02 5 86 3 40 12 13 31 14 04 12 07 11 52 13 23 25 32 52 32 93 37 43 14 30 11 21 35 26 21 30 63 15 20 17 9 89 10 86 9 42 16 4 62 2 72 2 41 1 57 17 1 12 0 29 0 52 0 52 18 0 28 t 19 D13S317 8 3 08 12 18 9 66 4 97 9 2 52 7 74 21 72 17 80 10 3 78 4 44 9 14 13 61 11 24 51 29 80 23 10 24 35 12 46 22 30 80 20 86 23 04 13 15 41 11 17 10 17 7 85 14 4 34 3 72 5 34 8 12 15 0 14 0 14 H 0 26 Experiments and Results 4 47 Table
14. 3 32 GeneScan Analysis software 3 30 information provided in the electropherogram 3 30 to 3 32 Ww WWW address Applied Biosystems A 5 Documents on Demand A 6 Index 3 Worldwide Sales Offices Applied Biosystems vast distribution and service network composed of highly trained support and applications personnel reaches into 150 countries on Six continents For international office locations please call our local office or refer to our web site at www appliedbiosystems com Headquarters 850 Lincoln Centre Drive Foster City CA 94404 USA Phone 1 650 638 5800 Toll Free 1 800 345 5224 Fax 1 650 638 5884 www appliedbiosystems com S AS BRIS Biosystems Applera Corporation is committed to providing the world s leading technology and information for life scientists Applera Corporation consists of the Applied Biosystems and Celera Genomics businesses Printed in the USA 05 2001 Part Number 4323291 Rev B
15. 5 to 3 11 setting up the instrument 3 4 to 3 5 using Performance Optimized Polymer 4 for analysis 3 2 to 3 34 DNA extraction D 2 to D 3 collecting and storing samples D 3 Q QuantiBlot Human DNA Quantitation Kit commonly asked questions E 5 to E 6 using for quantitation of human DNA E 2 using the kit E 3 to E 4 quantitation E 2 to E 6 commonly asked questions E 5 to E 6 QuantiBlot analysis E 3 to E 4 R results See viewing results S samples collecting and storing for extraction of DNA D 3 DNA from more than one individual 4 38 to 4 41 detecting 4 38 to 4 39 detection limit 4 40 to 4 41 resolving genotypes in mixed samples 4 40 to 4 41 setting up laboratories See laboratory setup STRBase 4 52 stutter peak or product 4 18 to 4 23 T tables allele frequencies of AmpF STR Identifiler loci 4 44 to 4 53 precision results from the two instrument platforms 4 10 to 4 15 troubleshooting B 1 to B 5 technical support A 1 to A 6 e mail address A 1 Internet address A 5 regional sales offices A 3 to A 5 telephone fax North America A 2 training obtaining information A 6 troubleshooting B 1 to B 5 automated genotyping 5 10 V validation of the AmpFZSTR Identifiler kit loci 4 2 kit reproducibility 4 6 minimum sample requirement 4 42 mixed specimen studies 4 42 mode of inheritance 4 27 optimizing PCR components 4 8 thermal cycler parameters 4 4 validation studies general considerations 4 2 to 4 36 viewing results 3 31 to
16. AmpFZSTR Identifiler Kit Template 5 10 Troubleshooting Automated Genotyping ssssnuusrnn 5 10 About This Kit Template 0000008 5 10 Categofles cuc Ue URN IRR be E Ae RUE 5 10 Offset Categories pel enti RIA 4 bed Cees 5 11 Off Ladder Alleles and Virtual Alleles 5 12 Kazam Macros ves e RR ERR ERA PI Rat qae Re t getan 5 13 Kazam 20 Filter 0 0 cece I 5 15 Modifying the Template 00 0200 2 eee eee 5 15 Determining Genotypes e cee eee eee 5 17 AmpF STR Identifiler Allelic Ladder 0000 5 17 Genotyping Using the AmpFZSTR Identifiler Allelic Ladder 5 19 A Technical Support Contacting Technical Support esee A 1 To Contact Technical Support by E Mail sees A 1 Hours for Telephone Technical Support A 2 To Contact Technical Support by Telephone or Pax A 2 To Reach Technical Support Through the Internet A 5 To Obtain Documents on Demand renerne A 6 To Obtain Customer Training Information A 6 B Troubleshooting OVELVICW 3 3 85 OUEST hs E bM ND eb PEL au B 1 About This Appendix 2 2 0 cece eee eee eee B 1 Ins Eis Appendixes a a ea festa aer A a ath hia RR a UR Re ee es B 1 Troubleshooune lt i 02 ue SZ eS eae us Hee eee EX Ween eh ROS B 2 C Laboratory Setup OV rVIeW uenire Sadie de ER EE E as C 1 About This Appendix ees C 1 In This Appendix eres ehe e wu RbURLORERVCRESDeB
17. Chapter This chapter describes the contents of the AmpFZSTRS Identifiler PCR Amplification Kit provides an overview of the kit and provides safety information This chapter contains the following topics Topic See Page Overview 1 1 Product Overview 1 2 Multicomponent Analysis Overview 1 3 Materials for the Kit 1 7 Safety 1 8 Introduction 1 1 Product Overview Purpose Five Dye DNA Fragment Analysis About the Primers 1 2 Introduction The AmpFLSTR Identifiler PCR Amplification Kit is a short tandem repeat STR multiplex assay that amplifies 15 tetranucleotide repeat loci and the Amelogenin gender determining marker in a single PCR amplification All thirteen of the required loci for the Combined DNA Index System CODIS loci are included in this kit for known offender databasing in the United States Budowle et al 1998a Two additional loci D2S1338 and D19S433 are included These loci are consistent with the AmpF STR SGM Plus PCR Amplification Kit The combination of the 15 loci are consistent with several worldwide database recommendations The Identifiler kit uses a five dye fluorescent system for automated DNA fragment analysis By adding an additional dye more loci can be multiplexed in a single PCR amplification as compared to the previous 4 dye system Applied Biosystems PET M and LIZ dyes expand the spectral detection range that can be used on ABI
18. Custom and Synthesis 1 800 899 5858 then press 1 then 5 1 508 383 7855 FMAT 8100 HTS System Cytofluor 4000 Fluorescence Plate Reader 1 800 899 5858 then press 1 then 6 1 508 383 7855 Chemiluminescence 1 800 542 2369 U S 1 781 275 8581 Tropix only or 1 781 271 0045 LC MS 1 800 952 4716 1 650 638 6223 Applied Biosystems MDS Sciex Outside North America Telephone Fax Region Dial Dial Africa and the Middle East Africa English Speaking and West Asia Fairlands South Africa 27 11 478 0411 27 11 478 0349 Africa French Speaking Courtaboeuf Cedex France 33 1 69 59 85 11 33 1 69 59 85 00 South Africa Johannesburg 27 11 478 0411 27 11 478 0349 Technical Support A 3 A 4 Technical Support Telephone Fax Region Dial Dial Middle Eastern Countries 39 0 39 8389 481 39 0 39 8389 493 and North Africa Monza Italia Easter n Asia China Oceania Australia Scoresby Victoria 61 3 9730 8600 61 3 9730 8799 China Beijing 86 10 64106608 or 86 10 64106617 86 800 8100497 Hong Kong 852 2756 6928 852 2756 6968 India New Delhi 91 11 653 3743 3744 91 11 653 3138 Korea Seoul 82 2 593 6470 6471 82 2 593 6472 Malaysia Petaling Jaya 60 3 79588268 603 79549043 Singapore 65 896 2168 65 896 2147 Taiwan Taipei Hsien 886 2 2358 2838 886 2 2358 28
19. PRISM genetic analysis instrumentation Together with 6 FAM VIC and NED dyes the spectral emission for this five dye set extends to 660 nm The AmpF4ZSTR Identifiler kit employs the same primer sequences as used in all previous AmpFZSTR kits A degenerate unlabeled primer for the D8S1179 locus was added to the AmpF STR Identifiler Primer Set in order to address a mutation observed in a population of Chamorros and Filipinos from Guam Budowle et a 1998b and Budowle et al 2000 The addition of the degenerate primer allows for the amplification of those alleles in samples containing this mutation without altering the overall performance of the AmpF STR Identifiler PCR Amplification Kit The data in this user s manual were generated prior to the addition of the degenerate primer Data showing equivalence with the degenerate primer will be published Non nucleotide linkers are used in primer synthesis for the following loci CSF1PO D281338 D138317 D168539 and TPOX For these primers non nucleotide linkers are placed between the primer and the fluorescent dye during oligonucleotide synthesis Grossman et al 1994 and Baron et al 1996 Non nucleotide linkers enable reproducible positioning of the alleles to facilitate inter locus spacing By combining the five dye system with the non nucleotide linkers for selected loci the same primer sequences developed for previous AmpFZSTR kits are used without modification Multic
20. Set G5 modules are installed and used for analysis Some but not all loci visible on electropherogram Test sample DNA is degraded If possible evaluate the quality of DNA sample by running an agarose gel If DNA is degraded re amplify with an increased amount of DNA Test sample contains PCR inhibitor e g heme compounds certain dyes Quantitate DNA and add minimum necessary volume Repeat test Wash the sample in a Centricon 100 Repeat test Troubleshooting B 3 Observation Possible Causes ABI PRISM 310 Genetic Analyzer Recommended Actions Data was not automatically analyzed Sample sheet not completed Complete sample sheet as described Injection list not completed Complete injection list as described Preferences not set correctly in ABI PRISM 310 Data Collection Software In Preferences under the Window menu select Injection List Defaults and the Autoanalyze checkbox Extra peaks visible when sample is known to contain DNA from a single source Incomplete denaturation before loading onto detection instrument Heat samples to 95 C for 3 min in deionized formamide solution Snap cool on ice Use Genetic Analyzer 0 5 mL Sample Tubes and a thermal cycler Current too high Decomposition of urea in the POP 4 polymer solution Add fresh POP 4 polymer solution to the syringe Incorrect buffer concentration Replace buffer with
21. allele 14 The reference size for this allele is 122 bp On a particular ABI PRISM 310 injection the size obtained for D381358 allele 14 was 119 06 bp The offset value is calculated as 119 06 122 2 94 In this example the actual category size used for allele assignment is 119 06 equals 122 2 94 which is the size of the D3S1358 allele 14 in this particular injection of the allelic ladder In other words the category sizes used for genotyping are equivalent to the allele sizes obtained in the lane or injection of allelic ladder Genotyping for the Macintosh OS 5 11 Off Ladder Alleles and Virtual Alleles Applying the Appropriate Offset Value to Each Allele in Succession Once the leftmost allele peak in each allelic ladder is identified the offset value determined for this allele is applied to the relevant allele s in the allele categories For example assume that the offset value determined by the 12 05 category in the D381358 os group is 3 01 for a particular lane or injection of allelic ladder This offset value is then applied to the allele 12 category in the D3S1358 group thus setting the correct offset value for allele 12 In order for the software to find the next allele peak in the D381358 allelic ladder allele 13 the offset value for the 12 os allele is also applied to the 13 0s category The result of this operation is that the 12 05 category size will be 4 bp longer than the 12 0s category In other word
22. amplification of loci High molecular weight DNA was incubated with the enzyme DNase for varying amounts of time The DNA was examined Experiments and Results 4 35 by agarose gel analysis to determine the average size of the DNA fragments at each time point Four nanograms of degraded DNA or 1 ng undegraded DNA was amplified using the AmpFZSTR Identifiler kit all 16 primer pairs together As the DNA became increasingly degraded the loci became undetectable according to size Preferential amplification was not observed The loci failed to robustly amplify in the order of decreasing size as the extent of degradation progressed CSF1PO and D281338 were the first loci to exhibit decreased amplification followed by D16S539 and D18S51 and so forth A similar result at each time point was obtained whether the DNA samples were amplified for each locus alone or co amplified with the AmpFZSTR Identifiler kit Figure 4 14 128 150 175 200 225 250 275 300 325 350 1680 1260 840 SUI LM M AANU J J JK NIA 1B A1 no DNase BE 1G Al no DNase UR 1v Al no DNase BE 18 41 no DNase 1680 1260 840 l LLAJA Mi M o A nA s BE 28 A220 seconds mE 26 A230 seconds OI 27 A230 seconds BR 25 12 20 seconds 1680 1260 840 420 o A A A A PTS 4B A4 1 minute BE 46 54 1 minute LIO 4Y A41 minutes BE 15 54 minute 1680 1280 840 420 oL A MA aA Aaa
23. are shown in Figure 4 16 where sample A and sample B were mixed according to the ratios provided The profiles of the samples in Figure 4 16 are the following Profile Allele Sample A Sample B Amelogenin X X Y CSF1PO 10 12 11 12 D281338 17 25 20 23 D381358 15 18 15 16 D5S818 11 13 11 D7S820 9 10 7 12 D8S1179 13 12 13 Experiments and Results 4 41 Profile Allele Sample A Sample B D138317 11 11 D168539 11 12 9 10 D18S51 17 19 12 15 D19S433 13 14 15 D21S11 30 30 2 28 31 FGA 23 2 24 24 26 THO1 7 9 7 9 3 TPOX 8 9 8 vWA 17 19 14 16 For these 1 ng total DNA mixture studies the limit of detection is when the minor component is present at approximately one tenth of the concentration of the major component and a threshold of 50 RFU The limit of detection for the minor component is influenced by the combination of genotypes in the mixture 1800 Panel 1 pn 600 o 1800 600 o EE 1800 3 1200 600 o 1800 4 1200 600 o 1800 5 1200 600 1800 6 1200 600 o 1800 7 1200 600 0 mm bl d silla AJL 9 n LL L n MN TRI Faily th Sample A 10 1 3 1 Sample B Figure 4 16 Results of the two DNA samples mixed together at defined ratios and amplified with the AmpFZSTR I
24. first lane or injection of ladder that is found is the one that is used by the Kazam macro in the AmpF STR Identifiler Kit Template to determine the sizes in the allele categories that will be used for genotyping It is possible to skip the first lane or injection of allelic ladder and use the second lane or injection of allelic ladder for genotyping instead After importing the sample files but before running the Kazam macro remove the word ladder from the Sample Info in all four sample dye colors for the first lane or injection of allelic ladder in the Dye lanes 5 2 Genotyping for the Macintosh OS window Make sure that the word ladder is entered for Sample Info in the second lane or injection of allelic ladder See step 3 on page 5 4 for a description of how to access the Sample Info field in the Dye lanes window GeneScan Analysis Software Peak Recognition All allele peaks in the allelic ladder for each locus must be recognized labeled in the GeneScan Analysis Software e each allele peak must have an entry in the GeneScan table Thus all allele peaks in each allelic ladder must have a peak height value in relative fluorescence units RFU that is greater than the Peak Amplitude Threshold PAT that was specified in the GeneScan software Analysis Parameters Also all allele peaks in each allelic ladder must be resolved For example the FGA 26 26 2 and 27 alleles must be resolved such that each peak has an entry in
25. from fresh or frozen whole blood peripheral blood lymphocytes blood stains sperm cells paraffin blocks teeth hair tissue bone and other biological samples There are numerous procedures that are currently being used for DNA extraction Some of these extraction procedures include Chelex phenol chloroform and FTA paper Regardless of the method used for DNA extraction all samples must be handled carefully to prevent sample to sample contamination or contamination by extraneous DNA Also when possible we recommend that the samples should be processed at a separate time from reference samples Phenol Chloroform Method The phenol chloroform method removes proteins and other cellular components from nucleic acids resulting in relatively purified DNA preparations This method results in double stranded DNA that is suitable for AmpFZSTR Identifiler kit amplifications DNA extracted by the phenol chloroform method is also suitable for RFLP analysis provided it is not significantly degraded This method is also recommended when extracting DNA from relatively large samples i e when the amount of DNA in a sample is expected to be greater than 100 ng Chelex Method The Chelex method of DNA extraction is more rapid than the phenol chloroform method It involves fewer steps resulting in fewer opportunities for sample to sample contamination This method produces single stranded DNA that is suitable for AmpFZSTR Identifiler kit amp
26. inhibitor of Taq DNA polymerase copurifies with human genomic DNA Nucleic Acids Hes 16 10355 References F 3 F 4 References DNA Advisory Board Federal Bureau of Investigation U S Department of Justice 1998 Quality assurance standards for forensic DNA testing laboratories DNA Recommendations 1994 Report concerning further recommendations of the DNA Commission of the ISFH regarding PCR based polymorphisms in STR short tandem repeat systems Intl J Legal Med 107 159 160 Edwards A Civitello A Hammond H and Caskey C 1991 DNA typing and genetic mapping with trimeric and tetrameric tandem repeats Am J Hum Genet 49 746 756 Edwards A Hammond H A Lin J Caskey C T and Chakraborty R 1992 Genetic variation at five trimeric and tetrameric tandem repeat loci in four human population groups Genomics 12 241 253 Fitzco Inc Product Bulletin Procedure for the processing of blood samples spotted on FTA paper for PCR amplification Maple Plain MN Frank W E and Llewellyn B E 1999 A time course study on STR profiles derived from human bone muscle and bone marrow J Forensic Sci 44 4 778 782 Fr geau C J and Fourney R M 1993 DNA typing with fluorescently tagged short tandem repeats a sensitive and accurate approach to human identification Biotechniques 15 100 119 Fregeau C J Leclair B Bowen K Elliott J Borys S and Fourney R 2000 The Swissair Flight 111 di
27. is calculated as described below peak A peak B x 100 percent stutter The percentage value that is used in the Genotyper software filtering option F can be derived from the conventional percent stutter expression S as follows F 10 000 S 100 For example if the desired stutter percent threshold for D381358 is 11 then the percentage value that should be used in the Genotyper software filtering option is F 10 000 11 100 835 To use a filter value different than 835 for D3S1358 simply input another value 5 Click Replace The peak filtering that is included in the Kazam macro is intended only as a tool and guideline Final conclusions should be based on careful examination of the STR profiles 5 14 Genotyping for the Macintosh OS Kazam The standard Kazam macro is written so that a different filter threshold 20 Filter Modifying the Template can be used for each locus the steps for each locus are written separately in the macro The Kazam macro thus provides maximum flexibility and the opportunity to customize the filter that is used for each locus A different version of the Kazam macro called Kazam 20 filter is also provided in the Macro list This macro is simpler than the Kazam macro in that a 20 stutter filtering step is specified for all loci To view the various steps in the Kazam 20 filter macro click on the Kazam 20 filter line in the Macro l
28. l di n La l Ji li i i Figure 3 7 Quick Tile Off and Quick Tile On options Panel A is an example of one sample displayed with Quick Tile Off Panels B F are examples of the same sample file with Quick Tile On using the AmpF4STR Control DNA 9947 analyzed on the ABI Prism 310 Genetic Analyzer 3 32 Note Eor a more detailed description see the GeneScan Software v3 1 User s Manual Standards for For the Identifiler kit the panel of standards needed for PCR Samples amplification PCR product base pair sizing and genotyping are as follows The AmpF STR Control DNA 9947A provides a positive control for the efficiency of the amplification step and STR genotyping using the AmpFZSTR Identifiler Allelic Ladder GeneScan 500 LIZ Size Standard is used for obtaining base pair sizing results The GeneScan 500 LIZ Size Standard is designed for sizing DNA fragments in the 35 500 bp range and contains 16 single stranded fragments of 35 50 75 100 139 150 160 200 250 not assigned when used on the ABI PRISM 310 Genetic Analyzer 300 340 350 400 450 490 and 500 bases This standard has been evaluated as an internal lane size standard and yields extremely precise sizing results of AmpFZSTR Identifiler kit PCR products The AmpFZSTR Identifiler Allelic Ladder was developed by Applied Biosystems for accurate characterization of the alleles amplified by t
29. liquid from the caps Select a clean unused tube for the master mix If you are preparing Then use a lt 84 samples and controls 1 5 mL microcentrifuge tube 85 110 samples and controls 2 0 mL microcentrifuge tube gt 110 samples and controls tube that is appropriate Calculate the required amount of components as shown Note The formulation in the list below provides a slight overfill to allow for volume lost in pipetting Number of samples X 10 5 uL of AmpFZSTR PCR Reaction Mix Number of samples X 0 5 uL of AmpliTaq Gold DNA Polymerase Number of samples X 5 5 uL of AmpFZSTR Identifiler Primer Set Vortex the master mix at medium speed for 5 sec Dispense 15 uL of master mix per PCR tube PCR Amplification 2 5 Preparing the DNA Samples DNA Sample Input Preparing the Samples 2 6 PCR Amplification DNA amplification with the AmpFZSTRe Identifiler kit requires 10 uL of DNA at a recommended concentration of 0 05 0 125 ng uL Note The final volume in each PCR tube is 25 uL To prepare the samples If you are preparing the Then DNA test sample tube and the sample DNA concentration is 0 125 ng uL Add 10 uL of sample to the PCR tube DNA test sample tube and the sample DNA concentration is 0 125 ng uL Dilute a portion of the sample with TE buffer see page 2 4 for preparation so that only 0 5 1 25 ng of total DNA is in a
30. not be incorrectly typed and will be identified as off ladder Any sample allele that sizes outside a window could be either of the following an off ladder allele i e an allele of a size that is not represented in the AmpFZSTR Identifiler Allelic Ladder anallele that does correspond to an allelic ladder allele but whose size is just outside a window because of measurement error The measurement error inherent in any sizing method can be defined by the degree of precision in sizing an allele multiple times Precision is measured by calculating the standard deviation in the size values obtained for an allele that is run in several lanes of one gel or in several injections in one capillary run Table 4 1 on page 4 10 indicates typical precision results obtained from the seven injections of the AmpFZSTR Identifiler Allelic Ladder analyzed on the ABI PRISM 310 Genetic Analyzer 47 cm capillary and POP 4 polymer The internal lane size standard used was GeneScan 500 LIZ These results were obtained within a set of injections on a single capillary As indicated above sample alleles may occasionally size outside of the 0 5 bp window for a respective allelic ladder allele because of measurement error The frequency of such an occurrence is lowest in detection systems having the smallest standard deviations in sizing Figure 4 3 on page 4 6 illustrates the tight clustering of allele sizes obtained on the ABI PRISM 310 Genetic Analyzer
31. on results 2 7 to 2 9 4 31 to 4 32 extraction protocols D 2 to D 3 collecting and storing samples D 3 how degraded DNA affects which loci amplify 4 34 to 4 35 mixed samples causing extra peaks 4 38 to 4 41 detecting 4 38 to 4 39 detection limit 4 40 to 4 41 resolving 4 39 to 4 41 quantitation E 2 to E 6 using agarose gel analysis to examine the DNA 4 35 Documents on Demand A 6 E effect of DNA quantity on results 2 7 to 2 9 effect of inhibitors 4 33 to 4 34 electropherogram Index 1 causes for extra peaks 4 40 to 4 41 addition of a 3 A nucleotide 4 24 DNA from more than one individual 4 38 to 4 41 stutter peak 4 18 to 4 23 viewing the results 3 30 to 3 32 e mail address for technical support A 1 evidence exclusion of suspects 4 43 extraction protocols D 2 to D 3 collecting and storing samples D 3 F Five Dye Analysis FTA cards using bloodstained FTA cards for amplification 2 9 1 2 to 1 5 G gels using an agarose gel to examine DNA 4 35 GeneScan Analysis software viewing results 3 30 GeneScan 500 LIZ Internal Lane Size Standard 3 22 3 30 4 7 5 19 to 5 20 genetics 4 43 to 4 52 allele frequencies 4 44 to 4 53 populations and samples used in studies 4 43 probability of identity 4 55 probability of paternity exclusion 4 56 genotype determining 4 6 AmpF STR Allelic Ladders 5 19 to 5 21 calculating precision data using the allelic ladders 4 7 to 4 17 exclusion of suspects 4 43 resolving in mixed sa
32. peak Look at the raw data and enter the values that will be appropriate for all sample files in the project These data points will affect what data is displayed in the results display b Data Processing The Baseline and the MultiComponent check boxes should be selected Choose a Smooth Option Smooth Options can affect peak height and peak definition The Light smoothing option is recommended for use with the AmpF4ZSTR products on the Macintosh computer c Peak Detection Choose a Peak Amplitude Threshold PAT for each dye color Use the active scroll bar to input the PATs for each of the five colors After analysis the GeneScan table will contain data for all peaks with a height above that specified by the PAT 3 26 Protocol for 310 Genetic Analyzer with Mac OS To set the analysis parameters continued Step Action Note We suggest that you determine the PATs appropriate for your analysis Sensitivity experiments should be conducted in your laboratory with each instrument to evaluate the PATs used for analysis The Min Peak Half Width for use with the AmpFZSTR products is 3 Pts d Size Call Range Choose the This Range Base Pairs radio button and enter the value of 75 for Min and 450 for Max e Size Calling Method Choose the Local Southern Method radio button for sizing of the AmpF STR products This method determines the sizes of fragments by using the re
33. peaks Heterozygote heterozygote one overlapping allele three peaks Heterozygote heterozygote two overlapping alleles two peaks Heterozygote homozygote no overlapping alleles three peaks Heterozygote homozygote overlapping allele two peaks Homozygote homozygote no overlapping alleles two peaks Z 9 9 9 9 9 Homozygote homozygote overlapping allele one peak Specific genotype combinations and input DNA ratios of the samples contained in a mixture determine whether it is possible to resolve the genotypes of the major and minor component s at a single locus The ability to obtain and compare quantitative values for the different allele peak heights on Applied Biosystems instruments provides additional valuable data to aid in resolving mixed genotypes This quantitative value is much less subjective than comparing relative intensities of bands on a stained gel Ultimately the likelihood that any sample is a mixture must be determined by the analyst in the context of each particular case including the information provided from known reference sample s Limit of Detection of the Minor Component Mixtures of two DNA samples were examined at various ratios 1 1 to 1 10 The total amount of genomic input DNA mixed at each ratio was 1 ng The samples were amplified in a GeneAmp PCR System 9700 and were electrophoresed and detected using an ABI PRISM 310 Genetic Analyzer The results of the mixed DNA samples
34. pump block below the syringe Manually press down on the 1 0 mL syringe plunger until the valve space is filled with polymer Note This will remove the air bubbles at this valve site and should use about 0 1 mL of polymer Tighten the waste valve to close To open the pin valve at the anode buffer reservoir on the pump block a From the Manual Control window select Buffer Valve Open b Click Execute Manually press down on the 1 0 mL syringe plunger to push enough gel through the block so that all of the air bubbles are removed from the polymer channel in the block This process should use about 0 2 mL of polymer IMPORTANT There should be no air bubbles in the pump block channels 10 a Close the pin valve by selecting Buffer Valve Close from the pop up menu in the Manual Control window b Click Execute 11 Move the syringe drive toggle to the right so that it is positioned over the syringe plunger 12 a From the Manual Control window select Syringe Down b Select 50 step intervals Execute until the toggle almost makes contact with the syringe plunger c Click Execute d Select smaller step intervals until the toggle makes contact with the syringe plunger 3 10 Protocol for 310 Genetic Analyzer with Mac OS Setting Up the Run Setting the Run Setting the run temperature prior to starting a run is optional however Temperature this step saves time This hea
35. shown in Figures 4 4 4 5 4 7 and 4 8 Smaller alleles display a lower level of stutter relative to the longer alleles within each locus This is reflected in Figures 4 4 through 4 7 where minimal data points are plotted for some smaller alleles as stutter could not be detected for many of these samples Forthe alleles within a particular locus the percent stutter is generally greater for the longer allele in a heterozygous sample this is related to the first point above Each allele within a locus displays percent stutter that is reproducible The highest percent stutter observed for each allele is as follows CSF1PO 9 296 D281338 11 196 D381358 10 7 D5S818 6 8 D7S820 8 2 D8S1179 8 2 D13S317 8 0 D16S539 Experiments and Results 4 19 4 20 Experiments and Results 10 4 D18851 17 0 D19S433 13 3 D21811 9 4 FGA 14 7 THO1 5 1 TPOX 4 8 and vWA 12 6 The highest observed percent stutter for each locus is included as the filtering step in Genotyper software Peaks in the stutter position that are above the highest observed percent stutter will not be filtered Peaks in the stutter position that have not been filtered and remain labeled can be further evaluated For evaluation of mixed samples see Mixed Samples The percent stutter does not change significantly with the recommended quantity of input DNA for on scale data The measurement of percent stutter may be unusually high for m
36. specificity such that it is specific to primates for the species tested with the exception of the Amelogenin locus The following experiments were conducted to investigate interpretation of AmpFZSTR Identifiler kit results from nonhuman DNA sources S Chimp daa st E dad L UW iad dd Sl Horse Lid i l L 2400 Pig B dil i l L L aol Dog A Ili i i i il J Em E coli o Lii l L i L L gun Negative Control i LL l L l Figure 4 11 Representative electropherograms of a primate non primates a microorganism and a negative control are shown All samples were analyzed on an ABI PRISM 310 Genetic Analyzer The peaks dipicted in orange are the GeneScan 500 LIZ size standard 4 30 Experiments and Results The extracted DNA samples were amplified in AmpFZSTR Identifiler kit reactions and analyzed using the ABI PRISM 310 Genetic Analyzer Primates gorilla chimpanzee orangutan and macaque 1 0 ng each Non primates mouse dog pig cat horse chicken and cow 2 5 ng each Bacteria and yeast Brochothrix Escherichia Neisseria Pseudomonas Bacillus Staphylococcus approximately 5 ng each and Saccharomyces 1 ng The primate DNA samples all amplified producing fragments within the 100 400 base pair region Lazaruk et al 2001 Wallin et a 1998 The microorganisms chicken cow cat and mouse did not yield detectable product Horse dog and pig produced a 103 bp fragment near the Amelogenin
37. template Step Action 2 Find the AmpF STR Identifiler Kit Template icon Click on the icon to select it From the File menu select Get Info a Deselect the check box for Stationery pad at the bottom of the window b Close the Get Info window Open the AmpF STR Identifiler Kit Template by double clicking on its icon Make any desired changes Save the template file by choosing Save from the File menu Repeat steps 2 and 3 o oj o a Select the check box for Stationery pad at the bottom of the window b Close the Get Info window 5 16 Genotyping for the Macintosh OS Determining Genotypes AmpF STR The AmpF STR Identifiler Allelic Ladder contains the most common Identifiler Allelic alleles for each locus Genotypes are assigned by comparing the sizes Ladder obtained for the unknown samples with the sizes obtained for the alleles in the allelic ladder Additional alleles have been included in the AmpFZSTR Identifiler Allelic Ladder for the FGA D18851 D21811 TH01 and vWA loci compared to those included for these same loci in other AmpFZSTR kits The macro size ranges include the actual number of nucleotides contained in the smallest and largest allelic ladder alleles for each locus as well as those alleles reported in STRBase www cstl nist gov div831 strbase as of September 2000 The size range also includes 3 A nucleotide addition and any shifts due
38. the AmpFZSTR Identifiler Allelic Ladder containing partial repeat units in population database and nonhuman primate DNA samples have been subjected to DNA sequencing at Applied Biosystems Lazaruk et al 2001 In addition other groups in the forensic community have sequenced alleles at some of these loci Nakahori et al 1991 Puers et al 1993 M ller et al 1994 Barber et al 1995 M ller and Brinkmann 1995 Barber et al 1996 Barber and Parkin 1996 Brinkmann et al 1998 Momhinweg et al 1998 Watson et al 1998 Among the various sources of sequence data on the AmpF4ZSTR Identifiler kit loci there is consensus on the repeat patterns and structure of the STRs Inheritance The AmpF STR loci have been validated by family studies to demonstrate their mode s of inheritance The Centre d Etude du Polymorphisme Humain CEPH has collected DNA from 39 families of Utah Mormon French Venezuelan and Amish descent These DNA sets have been extensively studied all over the world and are routinely used to characterize the mode of inheritance of various DNA loci Each family set contains three generations generally including four grandparents two parents and several offspring Consequently the CEPH family DNA sets are ideal for studying inheritance patterns Begovich et a 1992 Four CEPH family DNA sets were examined One and a half nanograms of DNA from each sample were amplified using the AmpF4STR SGM Plus kit followed
39. the ECL detection method Yes In fact a wise strategy is to perform a 15 minute film exposure first which gives sensitivity down to at least 150 pg Then place the film on the membrane for 3 hours or as long as overnight The longer exposure will give sensitivity down to about 20 pg The photon emission kinetics of ECL are such that many exposures can be taken in a relatively short period of time The light output is the greatest in the first hour gradually decreasing over the next several hours with a half life of about 60 minutes The results of one experiment for example indicated that six exposures could be taken in the first 2 5 hours of photon emission with each exposure detecting 80 150 pg of DNA A seventh exposure with the film on the membrane overnight was easily able to detect the 80 pg DNA sample Sometimes it is beneficial to perform a very short exposure about 5 minutes to facilitate quantitation of samples having intense signals in the range of 5 10 ng DNA DNA Quantitation E 5 Repeating the Can the probe be stripped off the membrane so that the QuantiBlot assay can be repeated if a mistake is made during the hybridization detection Assay steps Yes for the ECL method This procedure can be used with the TMB method only if no blue precipitate was deposited on the membrane The procedure is as follows Step Action 1 Heat 150 mL of the Wash Solution 1 5X SSPE 0 5 SDS to approximately 90
40. using PCR including reporting results of purchaser s activities for a fee or other commercial consideration is also granted with the purchase of said kit Further information on purchasing licenses to practice the PCR process may be obtained by contacting the Director of Licensing at Applied Biosystems 850 Lincoln Centre Drive Foster City California 94404 or at Roche Molecular Systems Inc 1145 Atlantic Avenue Alameda California 94501 Notice to Purchaser for Paternity Testing Use Limited License The purchase price of this product includes limited non transferable rights under U S Patents 4 683 202 4 683 195 and 4 965 188 or their foreign counterparts owned by Roche Molecular Systems Inc and F Hoffmann La Roche Ltd Roche to use only this amount of the product to practice the Polymerase Chain Reaction PCR Process described in said patents solely for the parentage determination testing applications of the purchaser and excludes analysis of forensic evidence The right to use this product to perform and to offer commercial services for parentage determination testing applications using PCR including reporting the results of the purchaser s activities for a fee or other commercial consideration is also hereby granted Further information on purchasing licenses to practice PCR and related processes may be obtained by contacting the Licensing Department Roche Molecular Systems Inc 1145 Atlantic Avenue Alameda California 94501
41. where the standard deviation in sizing is typically less than 0 15 bp The instance of a sample allele sizing outside of the 0 5 bp window because of measurement error is relatively rare when the standard deviation in sizing is approximately 0 15 bp or less Smith 1995 For sample alleles that do not size within a 0 5 bp window the PCR product must be rerun to distinguish between a true off ladder allele vs measurement error of a sample allele that corresponds with an allele in the allelic ladder Repeat analysis when necessary provides an added level of confidence to the final allele assignment Genotyper software automatically flags sample alleles that do not size within the prescribed window around an allelic ladder allele 4 8 Experiments and Results It is important to note that while the precision within a gel or set of capillary injections is very good the determined allele sizes vary between platforms Cross platform sizing differences arise from a number of parameters including type and concentration of polymer mixture run temperature and electrophoresis conditions Variations in sizing can also be found between runs on the same instrument and between runs on different instruments because of these parameters We strongly recommend that the allele sizes obtained be compared to the sizes obtained for known alleles in the AmpFZSTR Identifiler Allelic Ladder from the same run and then converted to genotypes as described in the
42. 0 049 0 044 D21S11 0 037 0 044 0 047 0 074 FGA 0 034 0 035 0 032 0 031 THO1 0 109 0 079 0 097 0 134 TPOX 0 089 0 188 0 168 0 159 vWA 0 066 0 066 0 080 0 103 Combined 1 31 x 10 18 5 01 x 10 18 7 65 x 10 18 3 62 x 10 17 The P value is the probability that two individuals selected at random will have an identical AmpFZSTR Identifiler kit genotype Sensabaugh 1982 The P values for the populations described in this section are then approximately 1 7 64 x 1017 African American 1 2 00 x 1017 U S Caucasian 1 1 31 x 1017 U S Hispanic and 1 2 76 x 1016 Native American 4 56 Experiments and Results Probability of Paternity Exclusion Table of Table 4 5 shows the Probability of Paternity Exclusion Pg values of the Probability of AmpFZSTR Identifiler kit STR loci individually and combined Paternity Exclusion Table 4 5 Probability of paternity exclusion for the AmpFZSTR Identifiler kit STR loci Locus African American U S Caucasian U S Hispanic Native American CSF1PO 0 545 0 496 0 450 0 409 D2S1338 0 748 0 725 0 671 0 399 D3S1358 0 591 0 630 0 495 0 510 D5S818 0 506 0 440 0 525 0 601 D7S820 0 591 0 582 0 574 0 492 D8S1179 0 580 0 680 0 599 0 601 D13S317 0 383 0 487 0 638 0 370 D16S539 0 649 0 566 0 567 0 428 D18S51 0 760 0 731 0 767 0 329 D19S433 0 601 0 531 0 678 0 360 D21S11 0 737 0 708 0 586 0 399 FGA 0 760 0 766 0 739 0 309 THO1 0 492 0 566 0 618 0 646 TPOX 0 521 0 329 0 392 0 687 vWA 0 709 0 625 0 555 0 5
43. 0 29 0 17 0 79 17 2 0 28 0 29 R 2 88 18 2 0 14 0 29 T 1 05 Experiments and Results 4 49 Table 4 3 AmpFZSTR Identifiler kit allele frequencies continued African American U S Caucasian U S Hispanic Native American Allele n 2 357 n 2 349 n 290 n 191 D21S11 24 24 2 0 14 0 43 0 17 S 24 3 0 28 y 25 25 2 i 0 14 0 17 9 26 0 14 0 14 0 17 D 27 5 04 4 58 1 21 0 52 28 22 97 16 76 9 14 6 28 28 2 29 19 33 20 49 21 21 16 75 29 2 0 14 T 0 52 0 26 29 3 0 14 5 ii 30 17 23 25 21 29 31 34 29 30 2 1 40 3 30 2 93 1 83 31 7 98 7 16 6 72 5 76 31 2 7 98 9 46 8 62 18 85 32 1 12 1 43 1 55 0 79 32 2 5 88 7 16 12 93 9 69 33 0 56 A 0 52 33 2 3 78 3 30 4 14 3 66 34 1 26 x x E 34 1 0 14 g R 34 2 0 14 0 29 0 86 0 79 35 2 94 E 0 34 N 35 1 0 14 K 5 a 35 2 2 0 14 36 0 84 d t 5 37 0 28 d j 38 0 14 x 4 50 Experiments and Results Table 4 3 AmpFZSTR Identifiler kit allele frequencies continued African American U S Caucasian U S Hispanic Native American Allele n 357 n 349 n 290 n 191 FGA 16 A 0 14 is 16 1 0 14 5 S 17 5 0 29 0 17 17 2 0 14 id 18 0 70 2 72 0 52 1 31 18 2 1 40 R x 9 19 6 72 6 16 7 07 10 21 19 2 0 28 N E 20 7 00 13 90 7 41 12 30 20 2 bi 0 14 4 21 12 89 16 91 14 66 12 83 21 2 ii 0 29 0 17 22 21 57 16 91 17 24 10 47 22 2 0 28 1 29 0 34 0 26 22 3 0 14 1 23 14 99 15 19 11 90 15
44. 0 mL glass distilled or deionized water CHEMICAL HAZARD EDTA may cause eye skin and respiratory tract irritation Please read the MSDS and follow the handling instructions Wear appropriate protective eye wear clothing and gloves Note Adjust the volumes accordingly for specific needs 2 Aliquot and autoclave the solutions 3 Store at room temperature Master Mix Prepare the master mix by combining AmpF STR PCR Reaction Mix AmpliTaq Gold9 DNA Polymerase and AmpFZSTRe Identifiler Primer Set reagents IMPORTANT The fluorescent dyes attached to the primers are light sensitive Protect the AmpF STR Identifiler Primer Set from light when not in use Also protect the AmpFZSTR Identifiler Allelic Ladder GeneScan 500 LIZ Size Standard and amplified fluorescently labeled PCR products from light 2 4 PCR Amplification To prepare the master mix Step Action 1 Determine the total number of samples including controls 2 IMPORTANT Vortex the following reagents for 5 sec AmpF STR PCR Reaction Mix AmpliTaq Gold DNA Polymerase AmpF STR Identifiler Primer Set px CHEMICAL HAZARD AmpliTaq Gold DNA Polymerase may cause eye and skin irritation It may cause discomfort if swallowed or inhaled Please read the MSDS and follow the handling instructions Wear appropriate protective eye wear clothing and gloves Spin the tubes briefly in a microcentrifuge to remove any
45. 1 and 12 differ by a single repeat unit 4 bp 5 12 Genotyping for the Macintosh OS Kazam Macro The D3S1358 group also contains virtual allele categories for other alleles such as 15 2 and 20 The offset value for allele 15 2 is specified to be the same as for allele 15 In this case since reference sizes for these two alleles differ by 2 bp the category size used for allele 15 2 will be 2 bp longer than for allele 15 Likewise the offset for allele 20 is specified to be the same as for allele 19 so the allele category size for allele 20 will be 4 bp longer than for allele 19 Many of the loci in the Categories window contain virtual allele categories For example the FGA locus contains a virtual category for many 2 bp length variants The Kazam macro is the top level macro that contains all of the instructions and steps necessary for determination of genotypes relative to the allelic ladder Kazam references the Calculate locus Offsets macros for each locus and contains further instructions to label peaks at each locus and to filter remove labels from the stutter peaks The various steps in Kazam can be viewed in the Genotyper software by clicking on the Kazam line in the Macro list and then choosing Show Step window under the View menu Filtering Stutter Peaks To illustrate the steps involved in filtering the stutter peaks consider again the example of the D381358 locus To filter stutter peaks Step Acti
46. 1X Genetic Analyzer Buffer No current No 1X Genetic Analyzer buffer Refill buffer vials with 1X Genetic Analyzer buffer Pump block channel blockage Remove and clean block Refer to the ABI PRISM 310 Genetic Analyzer User s Manual Loose valve fittings or syringe Tighten valve fittings and syringe Capillary not flush with electrode Tape capillary securely to heat plate Refer to the AB PRISM 310 Genetic Analyzer User s Manual P N 903565 Electrode bent Check calibration of autosampler No signal Capillary misaligned with electrode Align capillary and electrode No PCR product added Add 1 5 uL PCR product to formamide GeneScan 500 LIZ mixture Capillary bent out of sample tube Align capillary and electrode Recalibrate autosampler Autosampler not calibrated correctly Calibrate autosampler in X Y and Z directions PCR product not at bottom of tube Spin sample tube in microcentrifuge Air bubble at bottom of sample tube Spin tube in microcentrifuge to remove air bubbles Sealed sample tube septum Replace septum B 4 Troubleshooting Observation Low signal Possible Causes PCR product added to non deionized formamide Recommended Actions Always use deionized formamide for sample preparation Verify conductivity is lt 30 u siemens PCR product not mixed well with formamide GeneScan 500 LIZ mixture Mix PCR product with formamide Ge
47. 28 Combined 0 9999996 0 9999992 0 9999990 0 9999527 The Pz value is the probability averaged over all possible mother child pairs that a random alleged father will be excluded from paternity after DNA typing of the AmpFZSTR Identifiler kit STR loci Chakraborty and Stivers 1996 Experiments and Results 4 57 4 58 Experiments and Results Genotyping for the Macintosh OS Overview About This Chapter In This Chapter This chapter describes the use of ABI PRISMO Genotyper Software v2 5 2 in conjunction with the AmpFZSTR Identifiler Kit Template and the Macintosh OS to automatically genotype samples This chapter contains the following topics Topic See Page Overview 5 1 Using Genotyper Software for Automated Genotyping 5 2 Understanding the AmpFZSTR Identifiler Kit Template File 5 10 Determining Genotypes 5 17 Genotyping for the Macintosh OS 5 1 Using Genotyper Software for Automated Genotyping About the Software Before Running Genotyper Software Genotyper software is used to convert allele sizes obtained from ABI PRISM9 GeneScan Analysis Software into allele designations automatically and to build tables containing the genotype 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 A Genotyper software template file that contains macros specifically written for use wi
48. 39 Thailand Bangkok 66 2 719 6405 66 2 319 9788 Europe Austria Wien 43 0 1 867 35 750 43 0 1 867 35 75 11 Belgium 32 0 2 532 4484 32 0 2 582 1886 Czech Republic and 420 2 35365189 420 2 35364314 Slovakia Praha Denmark Naerum 45 45 58 60 00 45 45 58 60 01 Finland Espoo 358 0 9 251 24 250 358 0 9 251 24 243 France Paris 33 0 1 69 59 85 85 33 0 1 69 59 85 00 Germany Weiterstadt 49 0 6150 101 0 49 0 6150 101 101 Hungary Budapest 36 0 1 270 8398 36 0 1 270 8288 Italy Milano 39 0 39 83891 39 0 39 838 9492 Norway Oslo 47 23 12 06 05 47 23 12 05 75 Poland Lithuania Latvia 48 22 866 40 10 48 22 866 40 20 and Estonia Warszawa Portugal Lisboa 351 0 22 605 33 14 351 0 22 605 33 15 Russia Moskva 7 502 935 8888 7 502 564 8787 South East Europe Zagreb Croatia 385 1 34 91 927 838 385 1 34 91 840 Spain Tres Cantos 34 0 91 806 1210 34 0 91 806 1206 Sweden Stockholm 46 0 8 619 4400 46 0 8 619 4401 Switzerland Rotkreuz 41 0 41 799 7777 41 0 41 790 0676 To Reach Technical Support Through the Internet Region Telephone Dial Fax Dial The Netherlands Nieuwerkerk a d IJssel 31 0 180 392400 31 0 180 392409 or 31 0 180 392499 United Kingdom 44 0 1925 825650 44 0 1925 282502 Warrington Cheshire All other countries no
49. 4 3 AmpFZSTR Identifiler kit allele frequencies continued African American U S Caucasian U S Hispanic Native American Allele n 2 357 n 2 349 n 290 n 191 D16S539 5 8 3 22 1 72 1 72 0 79 9 19 05 10 46 9 31 12 30 10 10 92 5 59 15 69 15 45 11 31 51 31 95 30 17 30 89 12 18 77 30 23 29 48 27 75 13 14 85 16 76 11 55 10 73 14 1 54 3 01 2 07 2 09 15 0 14 0 29 E D18S51 7 9 0 14 3 i 10 0 28 0 86 0 52 0 79 10 2 0 14 9 A 11 0 28 1 15 1 21 12 7 00 13 90 10 34 14 92 13 4 34 12 18 14 48 9 16 13 2 0 42 N 14 6 86 16 76 15 52 26 96 14 2 0 28 E N 15 19 47 13 61 16 55 12 04 16 16 53 13 61 11 72 10 73 17 18 21 12 32 14 14 14 66 18 11 90 7 74 6 72 2 62 19 6 02 4 44 4 14 3 93 20 4 90 1 72 2 24 1 83 21 2 10 1 00 1 03 1 31 22 0 70 0 43 0 52 0 79 23 0 42 0 14 0 52 0 26 24 S 0 14 0 17 E 25 2 E 0 17 ui 26 27 4 48 Experiments and Results Table 4 3 AmpFZSTR Identifiler kit allele frequencies continued African American U S Caucasian U S Hispanic Native American Allele n 357 n 349 n 290 n 191 D19S433 9 N 0 14 0 17 i 10 1 54 E 11 7 14 0 72 0 52 0 52 11 2 0 14 0 17 12 10 78 7 74 6 21 3 14 12 2 6 30 0 57 1 90 13 29 83 28 94 16 03 17 80 13 2 5 74 1 72 8 62 15 45 14 21 01 34 10 31 72 24 87 14 2 4 20 0 86 5 00 3 66 15 4 76 15 76 13 45 13 35 15 2 3 36 2 72 8 79 10 73 16 2 38 4 15 4 31 3 93 16 2 2 38 1 72 2 93 1 83 17 A
50. 5 654 iu esu tee ERRA RE Pe ASL Thermal Cycler Parameters 0 0000 e eee ee eee AmpliTaq Gold DNA Polymerase Activation PCR Cycle Number eue ARR ceeds REEE LEE T Accuracy Precision and Reproducibility 0 020 000 20 eee 8 1 2 Accuracy xe eee p ep reeds SO Precision and Size Windows lee 4 7 Extra Peaks in the Electropherogram esee 4 18 Causes of Extra Peaks aar aa a ee eee ee 4 18 ATUfactst crie OE b E M E eee A tels 4 25 Characterization of Loci 4 27 8 1 2 1 Documentation e ee 4 27 OVERVIEW les Sys See bet ied igh EaR dT ARR T eere EUPeP d 4 27 Nature of the Polymorphisms 00 0 ee eee eeee 4 27 Inheritance erae ese MEE pee urs e ace ter Ee 4 27 Mappin gies oer pep ee oe bees 4 28 Species Specificity ice seed recoge E SER A ern VC ERES 4 29 8 1 2 2 Species Specificity eese 4 29 SENSIUIVILY e resnie sore gs ee TA ER A SRE RG AR ERER gees ewes T T 4 31 8 1 2 2 SensItvItyi re Sk otek ale ald bus Late eda a 4 31 Importance of Quantitation 0 002 202 eee 4 31 Stability se 5 ueste wees IR COE we PER SP Yea ees 4 33 8 11 22 Stability 2235 28 00 eek RIED REM WEA CREE 4 33 OVerVIeW ia oe ee RR RR ARORA daa R RRR R ETRE 4 33 Differential and Preferential Amplification 4 33 Effect of Inhibitors 4 33 Degraded DNA vo d ieu pe RR ELI EUR E ERES 4 34 Mixture Studies esL rep p EIS a a ee e eren 4 38 8 1 2 2 Mixture Studies llle 4 38 Mixed Specim
51. 55 63 0 05 13 159 81 0 06 14 164 04 0 07 15 167 95 0 05 16 172 09 0 05 D7S820 6 255 15 0 08 7 259 21 0 07 8 263 24 0 07 9 267 26 0 09 10 271 32 0 08 11 275 35 0 06 12 279 42 0 07 13 283 42 0 06 14 287 48 0 10 15 291 58 0 06 Experiments and Results 4 11 4 12 Table 4 1 Example of precision results of seven injections of the AmpF STR Identifiler Allelic Ladder continued ABI Prism 310 Genetic Analyzer Allele Mean S D D8S1179 8 123 29 0 07 9 127 32 0 05 10 131 41 0 05 11 135 49 0 04 12 139 73 0 04 13 144 25 0 03 14 148 71 0 06 15 153 16 0 07 16 157 51 0 07 17 161 72 0 05 18 165 84 0 07 19 169 92 0 05 D13S317 8 216 87 0 05 9 220 83 0 05 10 224 77 0 07 11 228 88 0 07 12 232 81 0 05 13 236 68 0 07 14 240 69 0 06 15 244 68 0 09 D16S539 5 252 37 0 08 8 264 30 0 07 9 268 32 0 08 10 272 32 0 06 11 276 37 0 07 12 280 37 0 09 13 284 34 0 07 14 288 44 0 09 15 292 51 0 07 Experiments and Results Table 4 1 Example of precision results of seven injections of the AmpF STR Identifiler Allelic Ladder continued ABI Prism 310 Genetic Analyzer Allele Mean S D D18S51 7 262 07 0 08 9 270 22 0 06 10 274 34 0 09 10 2 276 36 0 06 11 278 41 0 08 12 282 49 0 05 13 286 57 0 06 13 2 288 63 0 05 14 290 77 0 04 14 2 292 78 0 05 15 294 91 0 07 16 299 07 0 06 17 303 50 0 07 18 307 94 0 09 19 312 40 0 11 20 316 71 0 09 21 320 99 0 14 22 325 24 0 11 23 329 40 0 11 24 333 54 0 15
52. 59 24 50 21 55 33 51 18 14 71 22 49 18 45 15 45 19 6 72 8 31 7 07 4 71 20 1 96 1 15 1 38 1 05 21 0 28 0 17 0 26 22 0 28 x E 23 24 0 14 t A minimum allele frequency 0 796 for the African American database 0 796 for the U S Caucasian database 0 9 for the U S Hispanic database and 1 3 for the Native American database is suggested by the National Research Council in forensic calculations Analyzing the Four Databases Analysis across the four databases of 2274 total chromosomes per locus revealed the following number of different alleles 10 CSF1PO alleles 13 D2S1338 alleles at least 12 D3S1358 alleles 11 D58818 alleles 9 D7S820 alleles 11 D8S1179 alleles 8 D13S317 alleles 8 D16S539 alleles 20 D18S51 alleles 17 D19S433 alleles 26 D21S11 alleles 31 d FGA alleles 9 THO1 alleles 7 different TPOX alleles and 13 vWA alleles In addition to the alleles that were observed and recorded in the Applied Biosystems databases other known alleles have been published or reported to us by other laboratories see STRBase www cstl nist gov div831 strbase Low Frequency Alleles Some alleles of the AmpFZSTR Identifiler kit loci occur at a low frequency For these alleles a minimum frequency five divided by 2n where n equals the number of individuals in the database was Experiments and Results 4 53 assigned for the AmpFZSTR Identifiler kit African American U S Caucasian U S Hispanic and Nativ
53. 6 48 Li H Schmidt L Wei M H Hustad T Lerman M I Zbar B and Tory K 1993 Three tetranucleotide polymorphisms for loci D381352 D381358 D381359 Hum Mol Genet 2 1327 Liu C Y Takayanagi K Asamura H Ota M Fukushima H 2000 Study on ACTBP2 Mutations in Japanese Progress in Forensic Genetics 8 Eds G F Sensabaugh et al Elsevier Science pp 46 48 Luna L G ed Manual of Histologic Staining Methods of the Armed Forces Institute of Pathology McGraw Hill Book Co New York 1968 Magnuson V L Ally D S Nylund S J Karanjawala Z E Rayman J B Knapp J l Lowe A L Ghosh S and F S Collins 1996 Substrate nucleotide determined non templated addition of adenine by Taq DNA polymerase Implications for PCR based genotyping and cloning BioTechniques 21 700 709 Mancuso D J Tuley E A Westfield L A Worrall N K Shelton Inloes B B Sorace J M Alevy Y G and Sadler J E 1989 Structure of the gene for human von Willebrand factor J Biol Chem 264 19514 19527 Mansfield E S Robertson J M Vainer M Isenberg A R Frazier R R Ferguson K Chow S Harris D W Barker D L Gill P D Budowle B and McCord B R 1998 Analysis of multiplexed short STR systems using capillary array electrophoresis Electrophoresis 19 1 101 107 Meyer E Wiegand P Rand S Kuhlmann D Brack M and Brinkmann B 1995 Microsatellite polymorphisms re
54. 65 Walsh P S Fildes N J and Reynolds R 1996 Sequence analysis and characterization of stutter products at the tetranucleotide repeat locus vWA Nucleic Acids Res 24 2807 2812 Watson S Kelsey Z Webb R Evans J and Gill P 1998 The development of a third generation STR multiplex system TGM In Olaisen B Brinkmann B and Lincoln P J eds Progress in Forensic Genetics 7 Proceedings of the 17th International ISFH Congress Oslo 2 6 September 1997 Elsevier Amsterdam pp 192 194 References F 9 F 10 References Watson S Allsop R Foreman L Kelsey Z and Gill P 2001 Sequenced allelic ladders and population genetics of a new STR multiplex system Forensic Sci Int 115 3 207 217 Weber J and Wong C 1993 Mutation of human short tandem repeats Hum Mol Genet 2 1123 1128 Weir B S 1996 Genetic data analysis Il Sunderland MA Sinauer Associates Inc Weir B S 1992 Independence of VNTR alleles defined by fixed bins Genetics 130 873 887 Ziegle J S Su Y Corcoran K P Nie L Mayrand P E Hoff L B McBride L J Kronick M N and Diehl S R 1992 Application of automated DNA sizing technology for genotyping microsatellite loci Genomics 14 1026 1031 Index A A nucleotide addition by AmpliTaq Gold to 3 end of amplicon 4 24 ABI PRISM 310 Genetic Analyzer protocol 3 3 to 3 37 data analysis 3 26 to 3 29 ending the run 3 34 preparing and loading
55. 6B AG 4 minutes DE 66 464minutes 7 UR eY 464 minutes IM R AG 4 minutes 1680 1260 840 420 0 8B AB 8 minutes Ell 56 488 minutes x R sv 8 minutes ME SR cS e minutes Figure 4 14 Multiplex amplifications of a DNA sample in the absence of DNase and the sample incubated for 30 sec 1 min 4 min and 8 min with DNase l analyzed using the ABI PRISM 310 Genetic Analyzer Multiplex Amplifications DNA samples were amplified in 16 separate reactions containing primers for only one AmpFZSTR Identifiler kit locus singleplex and a 4 36 Experiments and Results reaction containing all primers for the AmpFZSTR Identifiler kit loci multiplex DNA used as PCR template consisted of a sample that had been degraded for 1 min with DNase l Amplified samples were analyzed using the ABI PRISM 310 Genetic Analyzer Similar results were obtained genotype and peak height whether the DNA samples were amplified for each locus alone or co amplified in the AmpFZSTR Identifiler kit reaction see Figure 4 15 on page 4 38 When degraded DNA is suspected to have compromised amplification of one or more loci the molecular weight of the DNA can be assessed by agarose gel analysis If the DNA is degraded to an average of 400 base pairs in size or less adding more DNA template to the AmpFZSTR Identifiler kit amplification reaction may help produce a typeable signal for the loci Adding more DNA to the amplification may provide more of t
56. 97 23 2 0 14 0 86 0 26 24 17 51 13 75 15 34 15 71 24 2 t 0 14 0 17 25 7 98 8 60 14 14 14 14 26 3 50 2 72 6 90 4 45 26 2 2 R R 0 52 27 1 82 0 72 2 41 0 79 28 1 40 0 14 0 69 0 52 29 0 56 1 t 30 30 2 0 14 B 31 2 A x 32 2 iS Z 33 2 x t E 34 2 0 14 N R 5 42 2 N ii 43 2 Y 2 x Experiments and Results 4 51 Table 4 3 AmpFZSTR Identifiler kit allele frequencies continued African American U S Caucasian U S Hispanic Native American Allele n 2 357 n 2 349 n 290 n 191 FGA con t 44 2 0 28 A M 45 2 i 0 26 46 2 0 14 i 47 2 E N 48 2 0 14 x T 50 2 x 3 P 51 2 9 i i THO1 4 5 0 28 0 43 0 17 6 11 06 20 49 22 76 20 68 7 42 86 21 78 33 62 43 98 8 20 73 11 46 8 45 5 24 8 3 E 0 14 12 32 16 19 14 14 6 28 9 3 11 62 29 08 20 34 23 56 10 0 98 0 43 0 52 0 26 11 13 3 0 14 N A TPOX 6 6 72 0 14 0 34 i 7 2 24 N 0 34 0 26 8 36 13 53 30 49 66 37 96 9 21 15 11 60 7 24 4 19 10 9 24 4 30 4 66 3 40 11 21 43 25 93 27 24 39 27 12 3 08 4 73 10 52 14 92 13 4 52 Experiments and Results Table 4 3 AmpFZSTR Identifiler kit allele frequencies continued African American U S Caucasian U S Hispanic Native American Allele n 357 n 349 n 290 n 191 vWA 11 0 28 2 0 17 9 12 0 26 13 1 26 0 43 ij 0 26 14 7 14 8 31 6 90 4 45 15 20 03 11 32 10 00 7 07 16 26 75 23 35 34 31 32 98 17 20
57. AmpF STR Identifiler PCR Amplification Kit User s Manual A Applied ns Copyright 2001 Applied Biosystems All rights reserved For Research Forensic and Paternity Use Only Not For Use in Diagnostic Procedures Printed in the U S A Notice to Purchaser for Research and Forensic Testing Use Limited License A license under U S Patents 4 683 202 4 683 195 and 4 965 188 or their foreign counterparts owned by Roche Molecular Systems Inc and F Hoffmann La Roche Ltd Roche for use in research and forensic testing has an up front fee component and a running royalty component The purchase price of the AmpFZSTR Identifiler PCR Amplification Kit P N 4322288 includes limited non transferable rights under the running royalty component to use only this amount of that product to practice the Polymerase Chain Reaction PCR and related processes described in said patents solely for the forensic testing and research activities of the purchaser when this product is used in conjunction with a thermal cycler whose use is covered by the up front fee component Rights to the up front fee component must be obtained by the end user in order to have a complete license These rights under the up front fee component may be purchased from Applied Biosystems or obtained by purchasing an Authorized Thermal Cycler The right to use the AmpFZSTR Identifiler PCR Amplification Kit to perform and to offer commercial services for forensic testing
58. Analysis could not be the GeneScan Parameters found software Analysis E Parameters b Reanalyze the sample file s containing the allelic ladder c Import all sample files into a new Genotyper software project and run the Kazam macro again About This Kit This section describes the organization and functionality of the Template AmpFZSTR Identifiler Kit Template Read this section for a greater understanding of the macros and steps that are used in the AmpFZSTR Identifiler Kit Template Categories In the Genotyper software each allele is defined by a category Each category contains information about the allele size size range and dye color To view the list of categories in the AmpFZSTR Identifiler Template choose Show Categories window under the View menu The categories for each locus are listed together under the locus name The locus is called a group In the Categories window each locus actually has two sets of categories For example the D3S1358 locus has one list of categories under the group D3S1358 and another list of categories under the 5 10 Genotyping for the Macintosh OS Offset Categories group D3S1358 os The categories in the D3S1358 group are allele categories and are used for allele assignment As noted above the offset values are determined automatically by the Calculate locus Offsets macros These macros use the offset categories categories with an os suffix to find the alle
59. Automated Genotyping section For more information on precision and genotyping see Lazaruk et al 1998 and Mansfield et al 1998 1187 population database DNA samples have been typed using the AmpFZSTR Identifiler PCR Amplification Kit see About the Primers on page 1 2 These samples have been previously genotyped with concordant results of the same loci using other AmpFZSTR kits Experiments and Results 4 9 Table 4 1 Example of precision results of seven injections of the AmpF4ZSTR Identifiler Allelic Ladder ABI Prism 310 Genetic Analyzer Allele Mean S D Amelogenin X 107 02 0 04 Y 112 61 0 02 CSF1PO 6 304 69 0 08 7 309 01 0 10 8 313 30 0 10 9 317 55 0 11 10 321 97 0 12 11 325 86 0 11 12 329 97 0 13 13 334 00 0 10 14 338 04 0 11 15 341 84 0 08 D2S1338 15 307 30 0 11 16 311 65 0 11 17 315 91 0 12 18 320 16 0 12 19 324 34 0 12 20 328 44 0 08 21 332 58 0 11 22 336 62 0 09 23 340 57 0 11 24 344 18 0 07 25 347 78 0 07 26 351 39 0 07 27 355 08 0 07 28 358 77 0 05 4 10 Experiments and Results Table 4 1 Example of precision results of seven injections of the AmpF STR Identifiler Allelic Ladder continued ABI Prism 310 Genetic Analyzer Allele Mean S D D3S1358 12 111 96 0 06 13 116 04 0 04 14 119 99 0 04 15 123 89 0 02 16 128 06 0 05 17 132 24 0 05 18 136 30 0 06 19 140 43 0 03 D5S818 7 134 14 0 05 8 138 21 0 04 9 142 56 0 04 10 147 02 0 06 11 151 31 0 01 12 1
60. DNA Analyzer 1 800 831 6844 then press 8 1 650 638 5981 Fluorescent Fragment Analysis includes GeneScan applications 1 800 831 6844 then press 2 then 3 1 650 638 5981 Integrated Thermal Cyclers ABI PRISM9877 and Catalyst 800 instruments 1 800 831 6844 then press 2 then 4 1 650 638 5981 ABI PRISM9 3100 Genetic Analyzer 1 800 831 6844 then press 2 then 6 1 650 638 5981 Peptide Synthesis 433 and 43X Systems 1 800 831 6844 then press 3 then 1 1 650 638 5981 Protein Sequencing Procise Protein Sequencing Systems 1 800 831 6844 then press 3 then 2 1 650 638 5981 PCR and Sequence Detection 1 800 762 4001 then press 1 for PCR 2 for the 7700 7900 or 5700 6 for the 6700 or dial 1 800 831 6844 then press 5 1 240 453 4613 Product or Product Area Telephone Dial Fax Dial Voyager MALDI TOF Biospectrometry Mariner ESI TOF Mass Spectrometry Workstations 1 800 899 5858 then press 1 then 3 1 508 383 7855 Biochromatography BioCAD Workstations and POROS Perfusion Chromatography Products 1 800 899 5858 then press 1 then 4 1 508 383 7855 Expedite Nucleic acid Synthesis Systems 1 800 899 5858 then press 1 then 5 1 508 383 7855 Peptide Synthesis Pioneer and 9050 Plus Peptide Synthesizers 1 800 899 5858 then press 1 then 5 1 508 383 7855 PNA
61. Estimation of mutation rates from parentage exclusion data applications to STR and VNTR loci Mutat Res 354 41 48 Chakraborty R Kimmel M Stivers D Davison L and Deka R 1997 Relative mutation rates at di tri and tetranucleotide microsatellite loci Proc Natl Acad Sci USA 94 1041 1046 Clark J M 1988 Novel non templated nucleotide addition reactions catalyzed by prokaryotic and eukaryotic DNA polymerases Nucleic Acids Res 16 9677 9686 Comey C T Koons B W Presley K W Smerick J B Sobieralski C A Stanley D M and Baechtel F S 1994 DNA extraction strategies for amplified fragment length polymorphism analysis J Forensic Sci 39 1254 1269 Cone R W and Fairfax M R 1993 Protocol for ultraviolet irradiation of surfaces to reduce PCR contamination PCR Methods Appl 3 815 817 Cotton E Allsop R Guest J Frazier R Koumi P Callow l Seager A and Sparkes R 2000 Validation of the AmpF STR SGM Plus system for use in forensic casework Forensic Sci Int 112 2 3 151 161 D2S1338 Cooperative Human Linkage Center CHLC accession number 41445 GenBank accession number G08202 D16S539 Cooperative Human Linkage Center CHLC accession number 715 GenBank accession number G07925 D19S433 Cooperative Human Linkage Center CHLC accession number 135 GenBank accession number G08036 DeFranchis R Cross N C P Foulkes N S and Cox T M 1988 A potent
62. P and Hormann S 2000 Modification of the stutter position label filtering macro in the PE Biosystems Genotyper Version 2 5 software package Resolution of stutter filter back talk Forensic Science Communications 2 3 References F 5 F 6 References Kwok S and Higuchi R 1989 Avoiding false positives with PCR Nature 339 237 238 Lazaruk K Walsh P S Oaks F Gilbert D Rosenblum B B Menchen S Scheibler D Wenz H M Holt C and Wallin J 1998 Genotyping of forensic short tandem repeat STR systems based on sizing precision in a capillary electrophoresis instrument Electrophoresis 19 1 86 93 Lazaruk K et al 2001 Sequence variation in humans and other primates at six short tandem repeat loci used in forensic identity testing Forensic Science International 119 1 1 12 Lareu M V Barral S Salas A Rodriguez M Pestoni C and Carracedo A 1998 Further exploration of new STRs of interest for forensic genetic analysis In Olaisen B Brinkmann B and Lincoln P J eds Progress in Forensic Genetics 7 Proceedings of the 17th International ISFH Congress Oslo 2 6 September 1997 Elsevier Amsterdam pp 192 200 Leclair B Fregeau C J Bowen K L Borys S B Elliott J and Fourney R M 2000 Enhanced kinship analysis and STR based DNA typing for human identification in mass disasters Progress in Forensic Genetics 8 Eds G F Sensabaugh et al Elsevier Science pp 4
63. PRISM 310 Genetic Analyzer Accessories ABI PRISM 310 Genetic Analyzer Capillary L 47 cm Lus 36 cm i d 50 um P N 402839 labeled with a green mark ABI PRISM 310 Genetic Analyzer Vials 4 0 ABI PRISM 310 Genetic Analyzer 0 5 mL Sample Tubes P N 401957 ABI PRISM 310 Genetic Analyzer Septa for 0 5 mL Sample Tubes P N 401956 Syringe Kloehn 1 0 mL P N 4304471 Benchkote absorbent protector sheets Flush cutting wire cutter P N T 6157 Freezer 15 to 25 C non frost free Gloves disposable powder free Glassware Ice bucket Kim Wipes Lab coat Microcentrifuge tubes 1 5 mL Microtube racks filter Nalgene filter apparatus 150 mL 0 2 um CN Permanent ink pen Pipette bulb Pipettes serological Protocol for 310 Genetic Analyzer with Mac OS 3 35 Equipment Required continued Equipment Pipette tips sterile disposable hydrophobic filter plugged Pipettors adjustable 1 10 uL 2 20 uL 20 200 uL and 200 1000 uL Refrigerator Repeat pipettor and Combitips that dispense 25 125 uL optional Sink Syringe 35 cc optional Tape Thermal cycler Tube 50 mL Falcon Tube decapper autoclavable Reagents Required Reagents Required Reagents ABI PRISM 310 10X Genetic Analyzer Buffer with EDTA P N 402824 AG501 X8 ion exchange resin Bio Ra
64. Primers ve iere n EIAS ERE RE AEN 1 2 Multicomponent Analysis Overview 0 02 c eee eee ee eee 1 3 About Multicomponent Analysis 0 000002 1 3 How Multicomponent Analysis Works esee 1 3 Loci Amplified by the Kit 0 2 2 cee eee eee 1 4 About This User s Manual 0 00 eese 1 6 Materials for the Kit eeu SS a RR RE eee eee 1 7 Kit Contents 2 0 bss de ote hho eb NAS 1 7 Kit Storage and Stability llle essere 1 7 SAL Ye ocho RER de rotes Pesce tet pue a eet en dee eic 1 8 Documentation User Attention Words 1 8 Chemical Hazard Warning e 2 cee ee eee 1 8 Site Preparation and Safety Guide eese 1 9 About MSDSS8 ilc ERI aa 1 9 Ordering MSDSs ie pu Re beer uer EE E E 1 10 2 PCR Amplification QYerVIeW otic SE eoa ee ERR VIR S Ie CER Aden tee 2 1 About This Chapter cn sae eR ee 2 1 In IhisCh pter o eite du 2255 wea CQ 2 1 PCR Work CG HR eger R N TRAR dS eae bende BEGG RETHARPTS 2 2 Setup Work Are ree mirra t miranti pea e pede qr a de 2 2 Amplified DNA Work Area seen 2 2 PCR Equipment and Materials 0 00 00 eee eee ee 2 3 Equipment and Materials Required But Not Supplied 2 3 Preparing the Reagents 0 0 eee eee eee 2 5 TE B roen eiee ace Skea de EN es ey Be ow EUER 2 5 Master Mixes ouai enge ed s EE ee 2 5 Preparing the DNA Samples e eese 2 7 DNA Sample Input sss e e e e e e e e 2 7 Preparing the Samples 00 0 02 c eee eee eee
65. RESNS C 1 Lab Design eor DIR eee V seu e ata t eme C 2 sensitivity of PER ciens ee eee we EAE ELO DAE C 2 D DNA Extraction Protocols 16 Cu 2h we Chee edd t pane Woe eke Ge D 1 About This Appendix ee D 1 In This Appendix rei e RR RR ERR tee eh oe als D 1 Introductions 32 cece A eR UC I REM E SC E Dt D 2 Overview of DNA Sample Types esee D 2 DNA Extraction Methods 00 000 e eee eee eee D 2 Warnings to Users 0 ee ee D 3 Collection and Storage of Samples for DNA Extraction D 3 Proper Collection eea RR ga bane ee rae EIER eens D 3 E DNA Quantitation OVERVIEW rece re eL RENS a p a Ga deu ees E 1 About This Appendix ee E 1 In This Appendix lt 62 Sod R careia i KRK Z RT Z E Za nee E 1 Importance of Quantitation eee E 2 DNA Quality aK 622000 dao bs Bi ee ee ee E 2 Quantitation and PCR Amplification 002 E 2 Using the QuantiBlot KIL 0 0 eee eee E 3 How the Kit Works 0 00 cee eee eee E 3 Specificity for Primate DNA 0 0 0 0 0 022 eee eee E 3 Commonly Asked Questions about the QuantiBlot Kit E 5 How Much DNA ntur ReheREIePLESPEEPWPDFeDSpes E 5 Multiple Film Exposures 0 00 0 e eee eee eee eee E 5 Repeating the Assay 0 0 00 RR eee eee eee eee E 6 Performing Hybridization and Detection at a Later Time E 6 F References Index Introduction Overview About This Chapter In This
66. Table 4 2 Peak Height Ratios Allele Number of Mean Median Minimum Maximum Observations n CSF1PO 84 86 88 63 6 99 8 D281338 93 84 86 42 8 99 7 D381358 91 88 90 64 3 99 7 D5S818 82 89 91 64 9 99 7 D7S820 96 89 90 66 2 99 5 D8S1179 89 90 93 57 5 99 8 D13S317 96 87 87 63 3 100 0 D16S539 92 88 91 61 5 99 9 D18S51 99 82 83 56 3 99 9 D19S433 98 88 92 48 8 100 0 D21S11 92 88 89 66 4 99 6 FGA 94 85 87 60 9 99 5 THO1 99 86 88 48 8 99 9 TPOX 87 87 92 55 9 99 8 vWA 101 86 88 62 8 99 1 Peak height ratios were determined for those heterozygous samples with peak heights gt 200 RFU For all 15 loci the mean peak height ratios indicate that the two alleles of a heterozygous individual are generally very well balanced If an unusually low peak height ratio is observed for one locus and there are no other indications that the sample is a mixture the sample may be reamplified and reanalyzed to determine if the imbalance is reproducible Possible causes of imbalance at a locus are degraded DNA presence of inhibitors extremely low amounts of input DNA or the presence of an allele containing a rare sequence that does not amplify as efficiently as the other allele Resolution of Genotypes in Mixed Samples A sample containing DNA from two sources can be comprised at a single locus of any of the seven genotype combinations listed below 4 40 Experiments and Results Heterozygote heterozygote no overlapping alleles four
67. VICTM NED PET and LIZ matrix standards run using the GS STR POP 4 1 mL G5 module Analysis Software This chapter was written for use with GeneScan Analysis 3 1 Software or higher Refer to the ABI PRISM 310 Genetic Analyzer User s Manual P N 903565 and GeneScan Analysis Software Version 3 1 User s Manual P N 403001 for more detailed information on the instrument and software used with these protocols Genotyper software v2 5 2 or higher must be used to analyze Identifiler kit data see Chapter 5 3 2 Protocol for 310 Genetic Analyzer with Mac OS Preparing the ABI PRISM 310 Genetic Analyzer ABI PRISM 310 Figure 3 1 shows the ABI PRISM 310 Genetic Analyzer The parts Genetic Analyzer mentioned in this section are labeled Refer to the ABI PRISM 310 Genetic Analyzer User s Manual P N 903565 and GeneScan Analysis Software Version 3 1 User s Manual P N 4306157 for more detailed information on the instrument and software used with this protocol Capillary Heat plate Syringe drive Syringe Anode buffer reservoir Pump block Autosampler X Electrode Figure 3 1 ABI PRISM 310 Genetic Analyzer Protocol for 310 Genetic Analyzer with Mac OS 3 3 Setting Up the Installing a New Electrode Instrument Installing and trimming a new electrode is usually necessar
68. ach matrix standard sample in this way see Figure 3 5 If this verification test fails then the capillary may not have been aligned properly in the instrument during the run Do the following a Repeat the experiment making sure that the capillary is placed carefully in the laser detection window b Tape the capillary to the heat plate so that the capillary is immobilized during the run Once a satisfactory matrix file has been made this matrix file can be applied to subsequent runs It is not necessary to run matrix standard samples for each new capillary Protocol for 310 Genetic Analyzer with Mac OS 3 21 Running DNA Samples Preparing Samples To prepare the samples and AmpF STR Identifiler Allelic S eP Action Ladder 1 Combine the necessary amount of Hi Di Formamide and GeneScan 500 LIZ Size Standard P N 4322682 in a single microcentrifuge tube as shown Number of samples 2 x 24 5 uL Hi Di Formamide Number of samples 2 x 0 5 uL GeneScan 500 LIZ Size Standard If you are using a multi channel pipettor or processing many samples you may want to prepare additional master mix CHEMICAL HAZARD Formamide is harmful if absorbed through the skin and may cause irritation to the eyes skin and respiratory tract It may cause damage to the central nervous system and the male and female reproductive systems and is a possible birth defect hazard Please read the MSDS and fo
69. ain peaks that are off scale The percent stutter for allele 15 in D381358 Figure 4 5 is artificially increased due to a reproducible artifact Figure 4 8 observed in the green dye lanes at this position When analyzing samples which contain a D3S1358 allele 15 we recommend careful examination due to the contribution that this identified artifact may add to the observed peak height or area The highest percent stutter for D381358 is not inconclusive of allele 15 Percent Stutter E a L L me e ame 9 TR 9 WO 9994 HO 9940 090 9 9999 ae 999 9 oe mam 090 000 99 0 oe as oe 9999 CCE 99 emu 9999 ma ove 9 belt oa oo ee ow 9 oo 9 m 9 00 99 aeo 9 w E o He Ri Z de dt TE 999 9 8 9 10 11 12 13 14 15 16 17 D8S1179 25 26 27 28 29 30 31 32 33 34 35 D21S11 Allele 8 9 10 11 12 13 14 D7S820 8 9 10 11 12 13 14 CSF1PO Figure 4 4 Stutter percentages for the D8S1179 D21S11 D78820 and CSF1PO loci Experiments and Results 4 21 S E 5 3 z o i o Z U eee o 3 N e i L ub Hb ui 3 L 333 ge gee oe E l i i l l 12 13 14 15 16 17 18 19 6 7 8 91011 8 9 10 11 12 13 14 8 9 10 11 12 13 14 16 17 18 19 20 21 22 23 24 25 26 D3S1358 THO1 D13S317 D16S539 D2S1338 Allele Figure 4 5 Stutter percentages for the D381358 THO1 D13S317 D16S539 and D2S1338 loci See the c
70. ak height threshold based on validation experiments performed in each laboratory to avoid typing when stochastic effects are likely to interfere with accurate interpretation of mixtures Evidence samples that contain body fluids and or tissues originating from more than one individual are an integral component of forensic casework Therefore it is essential to ensure that the DNA typing System is able to detect DNA mixtures In the case of STRs stutter peaks may be informative in the interpretation of mixed samples Furthermore alleles amplified with the AmpFZSTR Identifiler kit have similar peak height values for a heterozygous genotype within a locus This balance can be used as an aid in detecting and interpreting mixtures Detection of Mixed Samples Each of the following can aid in determining whether a sample is a mixture The presence of greater than two alleles at a locus The presence of a peak at a stutter position that is significantly greater in percentage than what is typically observed in a single source sample Significantly imbalanced alleles for a heterozygous genotype The peak height ratio is defined as the height of the lower peak in RFU divided by the height of the higher peak in RFU expressed as a percentage Mean median minimum and maximum peak height ratios observed for alleles in the AmpFZSTR Identifiler kit loci in unmixed population database samples are as follows Experiments and Results 4 39
71. ampened with distilled deionized water 2 Dry the electrode with a fresh Kim wipe tissue Note The autosampler should be recalibrated after cleaning the electrode as described in Calibrating the Autosampler of the ABI PRISM 310 Genetic Analyzer User s Manual Removing the Syringe To remove the syringe Step Action 1 Launch the ABI PRISM 310 Data Collection software 2 a Under the Window menu select Manual Control b Select Syringe Home from the Function pop up menu c Click Execute Note For all commands in the Manual Control window the Execute button must be selected to complete the task 3 Open the instrument doors and move the syringe drive toggle to the left 4 Unscrew the syringe from the pump block Checking the Syringe Verify that the 1 0 mL glass syringe P N 4304471 has a small O ring P N 221102 inside the syringe and that another O ring is placed around the ferrule shaped seal The ferrule should be firmly seated in the end of the 1 0 mL syringe If the syringe is dirty it must be cleaned before use Cleaning the Syringe To clean the syringe Step Action 1 Remove the plunger by slowly drawing it from the glass barrel count to 5 this should take approximately 5 sec while keeping the entire syringe submerged in water IMPORTANT Moving the dry plunger quickly can damage it resulting in premature failure or leakage around the plunger
72. ange of 0 5 1 25 ng of input DNA Adding greater than 1 25 ng of DNA can result in too much PCR product such that the dynamic range of the instrument used to detect and analyze the PCR product is exceeded See Off Scale Data on page 3 34 for more details Using the QuantiBlot Kit How the Kit Works Specificity for Primate DNA The method of DNA quantitation using the QuantiBlot Human DNA Quantitation Kit is based on probe hybridization to the human alpha satellite locus D17Z1 A biotinylated probe specific for the D17Z1 sequence is hybridized to sample DNA that has been immobilized via slot blot onto a nylon membrane The subsequent binding of horseradish peroxidase streptavidin enzyme conjugate HRP SA to the bound probe allows for either colorimetric or chemiluminescent detection In the case of colorimetric detection the oxidation of 3 35 5 5 tetramethylbenzidine TMB catalyzed by HRP SA results in the formation of a blue precipitate directly on the nylon membrane For chemiluminescent detection the oxidation of a luminol based reagent catalyzed by HRP SA results in the emission of photons that are detected on standard autoradiography film This process is called enhanced chemiluminescence ECL In both cases the quantity of sample DNA is determined by comparison of the sample signal intensity to human DNA standards that have been calibrated against two DNA controls of known quantity The colorimetric method allows fo
73. anufactured or distributed by Applied Biosystems call the chemical manufacturer 1 10 Introduction PCR Amplification Overview About This Chapter In This Chapter This chapter describes how to prepare the master mix for amplifying sample DNA using the AmpF STRS Identifiler PCR Amplification Kit prepare samples and controls and perform PCR This chapter contains the following topics Topic See Page Overview 2 1 PCR Work Areas 2 2 PCR Equipment and Materials 2 3 Preparing the Reagents 2 4 Preparing the DNA Samples 2 6 Performing PCR 2 7 PCR Amplification 2 1 PCR Work Areas Setup Work Area IMPORTANT These items should never leave the PCR Setup Work Area Amplified DNA Work Area 22 PCR Amplification 9 9 Z 9 9 9 9 Calculator Gloves disposable Marker pen permanent Microcentrifuge Microcentrifuge tubes 1 5 mL or 2 0 mL or other appropriate clean tube for Master Mix preparation Microcentrifuge tube rack Pipet tips sterile disposable hydrophobic filter plugged Pipettors Tube decapper autoclavable Vortex IMPORTANT The GeneAmp PCR Systems should be placed in the Amplified DNA Work Area Or GeneAmp PCR System 9700 GeneAmp PCR System 9600 PCR Equipment and Materials Equipment and Materials Required But Not Supplied The tables below list the equipment and materials required in addition to the reagents supplied with th
74. asses gloves or protective clothing For additional safety guidelines consult the MSDS Minimize the inhalation of chemicals Do not leave chemical containers open Use only with adequate ventilation e g fume hood For additional safety guidelines consult the MSDS Check regularly for chemical leaks or spills If a leak or spill occurs follow the manufacturer s cleanup procedures as recommended on the MSDS Comply with all local state provincial or national laws and regulations related to chemical storage handling and disposal Site Preparation and Safety Guide About MSDSs A site preparation and safety guide is a separate document sent to all customers who have purchased an Applied Biosystems instrument Refer to the guide written for your instrument for information on site preparation instrument safety chemical safety and waste profiles Some of the chemicals used with this instrument may be listed as hazardous by their manufacturer When hazards exist warnings are prominently displayed on the labels of all chemicals Chemical manufacturers supply a current MSDS before or with shipments of hazardous chemicals to new customers and with the first shipment of a hazardous chemical after an MSDS update MSDSs provide you with the safety information you need to store handle transport and dispose of the chemicals safely We strongly recommend that you replace the appropriate MSDS in your files each time you rece
75. ating OL Allele off ladder allele The Kazam macro includes a step that removes labels from stutter peaks by applying a percentage filter Labels are removed from peaks that are followed by a specified percent difference higher labeled peak within 3 25 to 4 75 bp The specified filter percentages for these loci are 186196 for THO1 1983 for TPOX 987 for CSF1PO 137196 for D58818 1150 for D13S317 835 for D381358 694 for vWA 580 for FGA 112096 for D8S1179 and D7S820 862 for D16S539 964 for D21S11 801 for D2S1338 652 for D19S433 and 488 for D18S51 These filter percentages correspond to the upper limit stutter percent values observed for each locus see Figures 4 4 through 4 7 Asample allele peak must have been recognized by GeneScan software before it can be recognized by Genotyper software Thus sample allele peaks that are below the PAT that was specified in the GeneScan software Analysis Parameters cannot be labeled by Genotyper software Also because no information is imported for peaks that are not recognized by GeneScan software such peaks will not align exactly by size relative to the x axis size scale in the Genotyper software plot window Peak Editing Clicking on a labeled peak removes the label Clicking again on the same peak defaults to the placement of bp size of that peak A dialog box with a field to enter the requested text may be accessed by Edit menu set click options Type t
76. atrix file none t Bd Autoanalyze with MAK Applied Biosystems Apps GeneScan Analysis Analysis Parameters file Analysis Default t Size Standard file none t C Auto Print 3 Make the following selections in the above window a Select GS STR POPA 1 mL G5 for the five dye module b Choose a default matrix file c Make sure the Genescan Analysis application is selected if you wish to autoanalyze If you do not wish to autoanalyze your data deselect the box next to the Autoanalyze with option Note When you create a new sample sheet a portion of the form is automatically filled in for you You can modify the automatic defaults in the Preferences file 4 Once you have finished making changes to the Preferences pages click OK to save your changes Running Matrix The precise spectral overlap between the five dyes is measured by 3 12 Samples analyzing DNA fragments labeled with each of the dyes 6 FAM VIC NED PET or LIZ dye in separate injections on a capillary These dye labeled DNA fragments are called matrix standard samples See Chapter 1 for a general description of multicomponent analysis The ABI PRISM GeneScan Analysis Software v3 1 or higher analyzes the data from each of these five samples and creates a matrix file The matrix file contains a table of numbers with five columns and five rows These numbers are normalized fluorescence intensities and r
77. by analysis using an 4 28 Experiments and Results ABI PRISM9 377 DNA Sequencer The families examined included 1331 11 offspring 13291 9 offspring 13292 9 offspring and 1113294 8 offspring representing 37 meiotic divisions The results confirmed that the loci are inherited according to Mendelian rules as has been reported in the literature Nakahori et a 1991 Edwards et al 1992 Kimpton et al 1992 Mills et a 1992 Sharma and Litt 1992 Li et al 1993 Straub et a 1993 Mapping The AmpFZSTR kit loci Amelogenin CSF1PO D2S1338 D3S1358 D58818 D78820 D8S1179 D13S317 D16S539 D18851 D19S433 D21811 FGA THO1 TPOX and vWA have been mapped and the chromosomal locations have been published Nakahori et a 1991 Edwards et al 1992 Kimpton et al 1992 Mills et a 1992 Sharma and Litt 1992 Li et a 1993 Straub et al 1993 Barber and Parkin 1996 Experiments and Results 4 29 Species Specificity 8 1 2 2 Species Species specificity sensitivity stability and mixture studies are conducted Specificity DAB 1998 The AmpFZSTR Identifiler kit provides the required degree of specificity such that it is specific to primates Other species do not amplify for the loci tested with the exception of the Amelogenin locus Nonhuman Studies Nonhuman DNA may be present in forensic casework samples The AmpF4ZSTR Identifiler kit provides the required degree of
78. c Analyzer Buffer b Insert the plastic vial lid with attached septum P N 402059 into the glass vial c Place the buffer vial into position 1 on the autosampler This will serve as the cathode buffer Note Overfilling and underfilling one or both buffer reservoir and vial can cause siphoning Pay close attention to the red fill line a Fill a second 4 mL glass buffer vial to the fill line with distilled water Insert the plastic vial lid with attached septum into the glass vial Place the vial into position 2 on the autosampler b C a Fill a 1 5 mL Eppendorf tube full with distilled water b Place it into position 3 on the autosampler Note Do not use a screw cap tube The lids on screw cap tubes are too high to clear the electrode and capillary Use a 1 5 mL Eppendorf tube with the lid clipped off Priming the Pump Block To prime the pump block Step Action 1 a From the Window menu select Manual Control b Select Buffer Valve Close from the pop up menu c Click Execute 2 Partly unscrew the capillary filling ferrule Protocol for 310 Genetic Analyzer with Mac OS 3 9 To prime the pump block continued Step Action 3 Manually press down on the 1 0 mL syringe plunger until the ferrule space is filled with polymer Note This will remove the air bubbles at the ferrule site Tighten the ferrule to close Partly unscrew the waste valve on the
79. ci obtained from relevant populations Where appropriate databases should be tested for independence expectations DAB 1998 To interpret the significance of a match between genetically typed samples itis necessary to know the population distribution of alleles at each locus in question If the genotype of the relevant evidence sample is different from the genotype of the suspect s reference sample then the suspect is excluded as the donor of the biological evidence tested An exclusion is independent of the frequency of the two genotypes in the population If the suspect and evidence samples have the same genotype then the suspect is included as a possible source of the evidence sample The probability that another unrelated individual would also match the evidence sample is estimated by the frequency of that genotype in the relevant population s The AmpFZSTR Identifiler PCR Amplification Kit prior to the addition of the D8S1179 degenerate primer was used to generate the population data provided in this section Samples were collected from individuals throughout the United States with no geographical preference African American 357 samples were provided by the Kentucky State Police and the Federal Bureau of Investigation U S Caucasian 349 samples were provided by the Kentucky State Police and the Federal Bureau of Investigation U S Hispanic 290 samples were provided by the Minnesota Bureau of Criminal Apprehe
80. ciprocal relationship between fragment length and mobility f Split Peak Correction Choose the None radio button no correction is needed for use with the AmpFZSTR products Click OK when done 5 Assign a size standard a Click the arrow in the Size Standard column for a sample file to view the pop up menu and select Define New For more information on defining a size standard refer to the GeneScan Analysis 3 1 User s Manual Do not assign a size for the 250 bp peak for data generated on the ABI PRISM9 310 Genetic Analyzer e assign a size of zero This peak can be used as an indicator of precision within a run Twelve size standard peaks should be viewed at this step as shown below AL wo a 150 m mo Es FS o 5 G0 S M6 s 5 1200 1000 200 s00 400 200 o Save the size standard for this sample in the GS Standards Folder within the ABI PRISM GeneScan Version 3 1 Software folder b To apply this standard to all injections choose the appropriate standard in the Size Standard column header above sample 1 in the Analysis Control window Protocol for 310 Genetic Analyzer with Mac OS 3 27 To set the analysis parameters continued Step Action 6 Analyze sample files a Highlight the blue green yellow red and orange columns Note Confirm that the orange box has been indicated as the standard a diamond symbol should app
81. ct technical support by e mail for help in the following product areas Product Area E mail address Genetic Analysis galab appliedbiosystems com Sequence Detection Systems and PCR pcrlab appliedbiosystems com Protein Sequencing Peptide and DNA Synthesis corelab appliedbiosystems com Biochromatography PerSeptive DNA PNA and Peptide Synthesis systems FMAT 8100 HTS System OytoFluor 4000 Fluorescence Plate Reader Voyager Mass Spectrometers Mariner Mass Spectrometers tsupport appliedbiosystems com Applied Biosystems MDS Sciex support sciex com Chemiluminescence Tropix tropix 9 appliedbiosystems com Technical Support A 1 Hours for Telephone Technical Support To Contact Technical Support by Telephone or Fax A 2 Technical Support In the United States and Canada technical support is available at the following times Product Hours Chemiluminescence 8 30 a m to 5 30 p m Eastern Time Framingham support 8 00 a m to 6 00 p m Eastern Time All Other Products 5 30 a m to 5 00 p m Pacific Time In North America To contact Applied Biosystems Technical Support use the telephone or fax numbers given below To open a service call for other support needs or in case of an emergency dial 1 800 831 6844 and press 1 Product or Product Area Telephone Dial Fax Dial ABI PRISM 3700
82. d Deionized water PCR grade Hi Di Formamide P N 4311320 GeneScan 500 LIZ Size Standard P N 4322682 Matrix Standard Set DS 33 6 FAM VIC NED PET LIZ for use with the 310 377 system P N 4318159 Performance Optimized Polymer 4 POP 4 P N 402838 3 36 Protocol for 310 Genetic Analyzer with Mac OS Software and User Documentation Software and User Documentation Required Software and User Documentation ABI PRISM 310 Collection Software version 2 1 or higher ABI PRISM 310 Genetic Analyzer User s Manual P N 903565 ABI PRISM 310 Module GS STR POP4 1 mL G5 GeneScan Software v3 1 or higher Protocol for 310 Genetic Analyzer with Mac OS 3 37 Experiments and Results Overview About This This chapter describes various experiments performed and results Chapter obtained using the AmpF STR Identifiler PCR Amplification Kit In This Chapter This chapter contains the following topics Topic See Page Overview 4 1 Experiments Performed Using the AmpFZSTR Identifiler PCR 4 2 Amplification Kit Developmental Validation 4 3 Accuracy Precision and Reproducibility 4 6 Extra Peaks in the Electropherogram 4 19 Characterization of Loci 4 28 Species Specificity 4 30 Sensitivity 4 32 Stability 4 34 Mixture Studies 4 39 Data Interpretation 4 43 Population Data 4 44 Mutation Rate 4 55 Probab
83. d due to the VIC dye artifact Note the degree of magnification y axis used in panels B and C to illustrate the artifact Data was produced on the ABI PRISM 310 Genetic Analyzer Addition of 3 A Nucleotide AmpliTaq Gold enzyme like many other DNA polymerases can catalyze the addition of a single nucleotide predominately adenosine to the 3 ends of double stranded PCR products Clark 1988 Magnuson et al 1996 This non template addition results in a PCR product that is one base pair longer than the actual target sequence and the PCR product with the extra nucleotide is referred to as the A form The efficiency of A addition is related to the particular sequence of the DNA at the 3 end of the PCR product The AmpFZSTR Identifiler kit includes two main design features that promote maximum A addition The primer sequences have been optimized to encourage A addition The final extension step is 60 C for 60 min This final extension step gives the AmpliTaq Gold DNA Polymerase extra time to complete A addition to all double stranded PCR product STR systems that have not been optimized for maximum A addition may have split peaks where each allele is represented by two peaks one base pair apart Experiments and Results 4 25 KA 170 175 190 185 199 195 No Extension A 300 200 100 9 lzel Paid 800 Final Extension c Figure 4 9 Split peaks resu
84. dentifiler PCR Amplification Kit Sample A and Sample B are a female and male sample respectively The ratios of Sample A to Sample B A B ratios shown are 10 1 3 1 1 1 1 3 and 1 10 respectively The alleles attributable to the minor component even when the major component shares an allele are highlighted in panels 2 3 5 and 6 All alleles are highlighted in panel 4 4 42 Experiments and Results Data Interpretation Minimum Sample The AmpFZSTR ldentifiler PCR Amplification Kit has been optimized to Requirement amplify and type approximately 0 5 1 25 ng of sample DNA reliably The PCR cycle number and amplification conditions have been specified to produce low peak heights for a sample containing 20 pg human genomic DNA Thus the overall sensitivity of the assay has been adjusted to avoid or minimize stochastic effects Applied Biosystems has successfully typed samples containing less than 0 5 ng DNA Note Individual laboratories may find it useful to determine an appropriate minimum peak height threshold based on their own results instruments using low amounts of input DNA Experiments and Results 4 43 Population Data 8 1 2 3 Population Data 8 1 2 3 1 Population Distribution Data Overview Population Samples Used in These Studies Population distribution data are documented and available DAB 1998 The population distribution data would include the allele and genotype distributions for the locus or lo
85. e American databases as suggested in the 1996 report of the Committee on DNA Forensic Science National Research Council 1996 These databases are summarized in Table 4 3 The minimum reportable genotype frequency at each locus is as follows 1 19 x 10 4 for the African American database 1 19 x 10 4 for the U S Caucasian database 1 70 x 10 4 for the U S Hispanic database and 2 97 x 10 4 for the Native American database p p 1 p 6 where 0 0 01 Hence the minimum combined multilocus genotype frequency at 15 loci is as follows 1 36 x 10 9 for the African American database 1 36 x 10 59 for the U S Caucasian database 2 86 x 10 5 for the U S Hispanic database and 1 23 x 10 53 for the Native American database 4 54 Experiments and Results Mutation Rate Estimating Germline Mutations Additional Mutation Studies Estimation of spontaneous or induced germline mutation at genetic loci may be achieved through comparison of the genotypes of offspring to those of their parents From such comparisons the number of observed mutations are counted directly In previous studies genotypes of ten STR loci amplified by the AmpF STR SGM Plus PCR Amplification Kit were determined for a total of 146 parent offspring allelic transfers meioses at the Forensic Science Service Birmingham England One length based STR mutation was observed at the D18S11 locus mutation was not detected at any of the other nine STR loci The D18S11 m
86. e AmpFZSTR Identifiler kit for PCR amplification Required Equipment Equipment Source GeneAmp9 PCR System 9700 Applied Biosystems P N N805 0001 Microcentrifuge Major laboratory supplier MLS Pipettors MLS Vortex MLS Required Materials Materials Source MicroAmp 96 Well Trays for Tubes with Caps Applied Biosystems P N N801 0541 MicroAmp Reaction Tubes with Caps 0 2 mL Applied Biosystems P N N801 0540 MicroAmp Reaction Tubes 8 tubes strip Applied Biosystems P N N801 0580 MicroAmp Caps 8 caps strip Applied Biosystems P N N801 0535 MicroAmp 96 Well Tray Retainer Set Applied Biosystems P N 403081 MicroAmp 96 Well Base Applied Biosystems P N N801 0531 MicroAmp Optical 96 Well Reaction Plate Applied Biosystems P N N801 0560 Microcentrifuge tubes 1 5 mL MLS Microcentrifuge tubes 2 0 mL MLS Pipet tips sterile disposable hydrophobic MLS filter plugged Tape labeling MLS Tube 50 mL Falcon MLS Tube decapper autoclavable MLS Deionized water PCR grade MLS Tris HCL pH 8 0 MLS 0 5 M EDTA MLS PCR Amplification 2 3 Preparing the Reagents TE Buffer The final concentration of TE buffer is 10 mM Tris HCI 0 1 mM EDTA pH 8 0 To prepare TE buffer Step Action 1 Mix together 10 mL of 1M Tris HCl pH 8 0 0 2 mL of 0 5 M EDTA 99
87. e been typed using the AmpFZSTR Identifiler PCR Amplification Kit These samples have been previously genotyped with concordant results of the same loci using other AmpFZSTR kits Peaks other than the target alleles may be detected on the electropherogram displays Several causes for the appearance of extra peaks including the stutter product found at the n 4 position incomplete 3 A nucleotide addition found at the n 1 position artifacts and mixed DNA samples see 8 1 2 2 Stutter Products The PCR amplification of tetranucleotide STR loci typically produces a minor product peak four bases shorter n 4 than the corresponding main allele peak This is referred to as the stutter peak or product Sequence analysis of stutter products at tetranucleotide STR loci has revealed that the stutter product is missing a single tetranucleotide core repeat unit relative to the main allele Walsh et a 1996 The proportion of the stutter product relative to the main allele percent stutter is measured by dividing the height of the stutter peak by the height of the main allele peak Such measurements have been made for amplified samples at the loci used in the AmpFZSTR Identifiler kit All data were generated on the ABI PRISM 310 Genetic Analyzer Some of the general conclusions from these measurements and observations are as follows For each AmpFZSTR Identifiler kit locus the percent stutter generally increases with allele length as
88. e information form if you have not previously done so then click Deliver Selected Documents Now to submit your order Note There is a limit of five documents per request for fax delivery but no limit on the number of documents you can order for e mail delivery To Obtain The Applied Biosystems Training web site at Customer www appliedbiosystems com techsupp training html Training provides course descriptions schedules and other training related Information information A 6 Technical Support Troubleshooting Overview About This Appendix In This Appendix Troubleshooting information addresses possible causes of a number of certain observations By following the recommended actions these observations described in this appendix may be further understood and or eliminated Appendix B contains the following topics Topic See Page Overview B 1 Troubleshooting B 2 Troubleshooting B 1 Troubleshooting Observation Possible Causes Recommended Actions Faint or no signal from both the AmpF STR Control DNA 9947A and the DNA test samples at all loci Incorrect volume or absence of either AmpF STR PCR Reaction Mix AmpF STR Identifiler Primer Set or AmpliTaq Gold DNA Polymerase Repeat amplification No activation of AmpliTaq Gold DNA Polymerase Repeat amplification making sure to hold reactions initially at 95 C for 11 min PCR Master Mi
89. ear in all orange boxes where a size standard is included with the sample files If the diamond symbol is not in the orange boxes mouse click will place a diamond in the box b Click the Analyze button After the analysis is complete confirm that the sizes for the peaks in the GeneScan 500 LIZ Size Standard have been correctly assigned a Open Results Control from the Window menu and examine the orange GeneScan 500 LIZ Size Standard peaks in overlapping groups of 16 samples Quick Tile Off Be sure to use the Align By Size option under the View menu b While the samples are tiled check the 250 bp peaks sized as approximately 246 bp in the enlarged view window Remember that this peak was not defined in the size standard The tiled 250 bp peaks should size consistently i e should all overlap In a typical run the 250 bp peaks all fall within a size window of approximately 1 bp Temperature fluctuations in the laboratory may cause variations 1 bp Note Laboratory temperature variations can cause size shifts If the temperature of the laboratory varies try injecting the AmpF STR Identifiler Allelic Ladder approximately every 10 injections or 5 hours c Scroll through the tables to verify correct GeneScan 500 LIZ peak assignments d Check the GeneScan 500 LIZ Size Standard peaks in the remaining samples taking note of which samples if any have incorrect peak assignments If the size standard peak a
90. eee 2 7 Performing PCR s cere ERI SH e ITA 2 8 Thermal CyclerS osipe Z SE A RL che aa EE REIR ate 2 8 Amplifying the DNA 0 0 00 eee ee eee 2 8 Amplification Using Bloodstained FTA Cards 2 9 Protocol for 310 Genetic Analyzer with Mac OS OverVIeWis ous ove ete P eae Re oy ee Pk ed cv Aes 3 1 About Lhis Chapter uixosievce se eR ER ee eee eee sie NSS 3 1 In This Chapter i aT a ea aa eat AS Guest ERa EE 3 1 Software Requirements e 3 2 Collection Software llle 3 2 Analysis Software Re E 6 a R cece ee ene 3 2 Preparing the ABI PRISM 310 Genetic Analyzer esses 3 3 ABI PRISM 310 Genetic Analyzer 0 00 00 00005 3 3 Setting Up the Instrument 0 00 0 00008 3 4 Setting Upithe Runes scc ee eases Y pEeDeEER E eDETeUR ER 3 11 Setting the Run Temperature 000000 3 11 Setting the Parameters llle 3 11 Running Matrix Samples esee 3 12 Filter Set GS Module Files 0 0 00 0c eee eee eee 3 14 OVEDVIEW Gie eo rick bce ited Gag eg ashe Sacer tga 3 14 Five Dye Data Collectio isons sre rii cene RR RE E RRR eee 3 15 OV erVIeW esi E eet un EGR Ooi Kane gene bates 3 15 Creating a Five Dye Sample Sheet and Injection List 3 15 Making a Matrix File s gas aaa RI RE RR E 3 18 Matrix Standards 0 0 0 eee eee eee 3 18 Making a Matrix File on the ABI PRISM 310 3 18 Running DNA Samples eee 3 22 Preparing Samples and AmpFZSTR Identifiler Allelic Ladde
91. en Studies eese lees 4 38 Data Interpretation xR R a der KR d R d Rr d e 4 42 Minimum Sample Requirement e esee 4 42 Population Data c scs ec epee eee awa a 4 43 8 1 2 3 Population Data 4 43 8 1 2 3 1 Population Distribution Data uus asssssaassaa 4 43 OVetrVIeW x s edet ue C e Ee dede saya e o E RE ios 4 43 Population Samples Used in These Studies suus 4 43 AmpFZSTR Identifiler Kit Allele Frequencies 4 44 Mutation Rate ove xed ee ae Ge ee ce edie qup ec spe 4 54 Estimating Germline Mutations 0 00008 4 54 Additional Mutation Studies 0 00 0 00 000 4 54 Probability of Identity 0 0 eee 4 55 Table of Probability of Identity 0 004 4 55 Probability of Paternity Exclusion 0 000 002 eee eee 4 56 Table of Probability of Paternity Exclusion 4 56 5 Genotyping for the Macintosh OS OVERVIEW 1s cerea ak do vast aad EIU ER RR CN EE CARE Ps 5 1 About This Chapter 0 00 0 0 00 eee eee eee eee 5 1 In This Chaptet e he ERU Eee ed ee be ee 5 1 Using Genotyper Software for Automated Genotyping 5 2 About the Software 0 gE RER eee eee eee 5 2 Before Running Genotyper Software 5 2 AmpFZSTR Identifiler Kit Template 5 3 Using the AmpFZSTR Identifiler Kit Template File 5 4 Examining Data sccis sl eer RE RR RE 5 6 Making Tables a R UE ion eae eae 5 7 Understanding the
92. epresent a mathematical description of the spectral overlap that is observed between the five dyes Figure 3 3 on page 3 13 The rows in the matrix file table represent the virtual filters and the columns represent the dye labeled DNA fragments indicated as Protocol for 310 Genetic Analyzer with Mac OS Reactions in Figure 3 4 on page 3 19 The top left hand value 1 0000 represents the normalized fluorescence of blue 6 FAM labeled DNA fragments in the blue filter It follows that all matrix tables should have values of 1 0000 on the diagonal from top left to bottom right as shown in Figure 3 3 D 310 G5 Matrix E Reactions B G Y R 0 Figure 3 3 Matrix file table indicating the values obtained on a particular ABI PRISM 310 System The values obtained will be unique for each instrument The other values in Figure 3 3 should all be less than 1 0000 These values represent the amount of spectral overlap observed for each dye in each virtual filter For example the values in the first column reflect quantitatively the amount of blue dye detected in each virtual filter These matrix file values will vary between different instruments virtual filter sets and run conditions on a single instrument A matrix file must be made for each instrument and for a particular set of run conditions The appropriate matrix file can be applied to data on subsequent runs on the same instrument as long as the
93. er alleles on the ABI PRISM 310 Genetic Analyzer with POP 4 polymer The x axis in Figure 4 5 represents the nominal base pair sizes for the AmpFZSTR Identifiler Allelic Ladder and the dashed lines parallel to the x axis represent the 0 5 bp windows The y axis is the deviation of each sample allele size from the corresponding allelic ladder allele size The data include a total of 2269 alleles from 70 population database samples All sample alleles are within 0 5 bp of a corresponding allele in an allelic ladder OD8s1179 AD21S11 XD75820 OCSF1PO XD35135B ATHOI OD138317 00165539 XD251338 30195433 OvWA D TPOX ADIBSSI XAMEL ADSSBIB TFGA 75 125 175 225 275 325 375 Allele Size bp Figure 4 3 Size deviation of 70 samples and two allelic ladders from one injection of allelic ladder on a single ABI PRISM 310 Genetic Analyzer run 4 6 Experiments and Results Experiments and Results 4 7 Precision and Size Sizing precision allows for determining accurate and reliable genotypes Windows Sizing precision was measured on the ABI PRISM 310 Genetic Analyzer As indicated in the Automated Genotyping section the recommended method for genotyping is to employ a 0 5 bp window around the size obtained for each allele in the AmpFZSTR Identifiler Allelic Ladder A 0 5 bp window allows for the detection and correct assignment of alleles An allele that sizes only one base pair different from an allele in the allelic ladder will
94. er kit contains sufficient quantities of the following reagents and the appropriate licenses to perform 200 25 uL amplifications Component Description Volume AmpF4STR9 PCR Two tubes containing MgCl 1 1 mL tube Reaction Mix deoxynucleotide triphosphates and bovine serum albumin in buffer with 0 05 sodium azide AmpF4STR One tube containing fluorescently 1 1 mL Identifiler Primer labeled primers and non labeled Set primers AmpliTaq Gold9 Two tubes of enzyme with an activity 50 uL tube DNA Polymerase of 5 U uL AmpF4STR9 One tube containing 0 10 ng uL 0 3 mL Control DNA human female cell line DNA in 9947A 0 05 sodium azide and buffer refer to pages 1 4 and 1 5 for profile AmpFL STR One tube of AmpF STR Identifiler 50 uL Identifiler Allelic Allelic Ladder containing amplified Ladder alleles See the table on pages 1 4 and 1 5 for a list of alleles included in the allelic ladder Kit Storage and The table below lists the storage temperature for the kit components Stability IMPORTANT The fluorescent dyes attached to the primers are light sensitive Protect the AmpFZSTR Identifiler Primer Set from light when not in use Amplified DNA AmpF STR Identifiler Allelic Ladder and GeneScan 500 LIZ Size Standard should also be protected from light Storage Component Temperature AmpF4STR PCR Reaction Mix 210 8 C AmpF4STR Identifiler Primer Set AmpF4STR Con
95. er the sample names and numbers for each injection in the Sample Name column c Using copy feature under Edit menu copy all sample names at one time by highlighting the Sample Name header and paste by highlighting the Sample Info header The sample name will appear in the blue green yellow red and orange Sample Info column for each sample From the File menu select New and click on the GeneScan Injection List icon Choose the appropriate sample sheet from the Sample Sheet pop up menu at the top left of the Injection List window 3 24 Protocol for 310 Genetic Analyzer with Mac OS To run the samples continued Step Action 6 From the Module pop up menu choose Module GS STR POP4 1 mL G5 for every injection a Click the arrow in the Module column for the first sample injection to view the pop up menu and choose the GS STR POP4 1 mL G5 module file b Select the entire Module column by clicking the Module column heading and choosing Fill Down from the Edit menu Note This function will not need to be performed if the preferences were set as described on page 4 12 From the Matrix file pop up menu choose the appropriate matrix file for every injection a Click the arrow in the Matrix column for the first sample injection to view the pop up menu and choose the appropriate matrix file Select the entire Matrix column by clicking the Matrix column heading and choosing Fil
96. es R Kimpton C Gilbard S Carne P Andersen J Oldroyd N Thomas D Urquhart A and Gill P 1996 The validation of a 7 locus multiplex STR test for use in forensic casework II Artefacts casework studies and success rates Int J Legal Med 109 195 204 Sparkes R Kimpton C Watson S Oldroyd N Clayton T Barnett L Arnold J Thompson C Hale R Chapman J Urquhart A and Gill P 1996 The validation of a 7 locus multiplex STR test for use in forensic casework I Mixtures ageing degradation and species studies Int J Legal Med 109 186 194 Straub R E Speer M C Luo Y Rojas K Overhauser J Ott J and Gilliam T C 1993 A microsatellite genetic linkage map of human chromosome 18 Genomics 15 48 56 Sullivan K M Mannucci A Kimpton C P and Gill P 1993 A rapid and quantitative DNA sex test fluorescence based PCR analysis of X Y homologous gene amelogenin Biotechniques 15 636 641 Sweet D and Hildebrand D 1999 Saliva from cheese bite yields DNA profile of burglar A case report Int J Legal Med 112 3 201 203 Szibor R Lautsch S Plate l Bender K and Krause D 1998 Population genetic data of the STR HumD381358 in two regions of Germany Int J Legal Med 111 160 161 Tanaka M Yoshimoto T Nozawa H Ohtaki H Kato Y Sato K Yamamoto T Tamaki K and Katsumata Y 2000 Usefulness of a toothbrush as a source of evidentia
97. gnal for any peak at a locus is greater than 8191 RFU the text 310 off scale will appear in this column Edited Label The text Edited will appear in this column for any loci where the peak labels were edited manually For example clicking on an unlabeled peak in the Plot window to add a label constitutes an edit Edited Row The text Edited will appear in this column for any rows in the table that contain table cells that have been edited after initial creation of the table IMPORTANT Before making a table all electropherograms should be examined and their peaks edited as described in the previous section To create and use tables Step Action 1 From the Macro list at the bottom of the Genotyper software Main Window click on one of the three table macros 2 From the Macro menu choose Run Macro 5 8 Genotyping for the Macintosh OS To create and use tables continued Step Action 3 Under the Views menu select Show Table Window to view the table in full screen mode For all tables except the Make Allele Table Clicking in a cell of the table causes the corresponding sample electropherogram to appear in the plot window as follows a Click on any cell in the table This causes this locus region of the corresponding electropherogram to appear in the Plot window for that sample b Zoomout H to view all loci for a particular dye color for the corresponding sam
98. gnation for each allele is shown in Figure 5 1 120 180 280 300 320 340 360 200 220 240 260 D21511 D75820 CSF1PO mm uu Dau pm 140 160 D881179 EI I CN EDI E D381358 THO1 D138317 0168539 D281338 1000 G Ta ee E e ose st ut 13 15 17 19 5 11 11 13 15 16 18 20 22 24 26 28 10 D198433 VWA TPOX D18551 pe juu i Co lta A p eeee Amelogenin Dpssgig SES LRS 1000 Figure 5 1 Genotyper software plot of the AmpFZSTR Identifiler Allelic Ladder indicating the designation for each allele These results were obtained on an ABI PRISM 310 Genetic Analyzer 5 18 Genotyping for the Macintosh OS Genotyping Using the AmpF STR Identifiler Allelic Ladder When interpreting AmpFZSTR Identifiler kit results genotypes are assigned to sample alleles by comparison of their sizes to those obtained for the known alleles in the AmpFZSTR Identifiler Allelic Ladder Genotypes not sizes are used for comparison of data between runs instruments and laboratories We strongly recommend that laboratories use an AmpF STR Identifiler Allelic Ladder from each project to convert the allele sizes to genotypes The main reasons for this approach are The size values obtained for the same sample can differ between instrument platforms because of differences in the type and concentration of the gel polymer matrices and in electrophoretic conditio
99. han two alleles present at a locus Possible Causes Quantity of test DNA sample is below assay sensitivity Recommended Actions Quantitate DNA and add 0 5 1 25 ng of DNA Repeat test Test sample contains PCR inhibitor e g heme compounds certain dyes Quantitate DNA and add minimum necessary volume Repeat test Wash the sample in a Centricon 100 Repeat test Test sample DNA is degraded If possible evaluate the quality of DNA sample by running an agarose gel If DNA is degraded re amplify with an increased amount of DNA Dilution of test sample DNA in HO or wrong buffer e g wrong EDTA concentration Presence of exogenous DNA Re dilute DNA using TE Buffer with 0 1 mM EDTA Use appropriate techniques to avoid introducing foreign DNA during laboratory handling Too much DNA in reaction Use recommended amount of template DNA 0 5 1 25 ng Mixed sample Amplification of stutter product n 4 bp position See Chapter 4 Experiments and Results Incomplete 3 A base addition n 1 bp position See Chapter 4 Experiments and Results Be sure to include the final extension step of 60 C for 60 min in the PCR Signal exceeds dynamic range of instrument off scale data Poor spectral separation bad matrix Quantitate DNA and re amplify sample adding 0 5 1 25 ng of DNA Follow the steps for creating a matrix file Confirm that Filter
100. have zeros in the cells of the table for that locus Loci that have homozygous alleles will have the allele designation indicated twice in the table Genotyping for the Macintosh OS 5 7 Make Allele Table This table has Sample Info in the first column and allele designations for each locus in columns 2 23 The first two labeled peaks within each locus appear in the table Make CODIS Table This table has Sample Info in the first column locus name in the second column and allele designations in columns 3 4 This alternative table is to facilitate laboratories that will be importing data into the Combined DNA Index System CODIS 310 Make Table This table can be used if the data was generated on the ABI PRISM 310 Genetic Analyzer This table has Sample Info in the first column Sample Comment in the second column locus name in the third column and allele designations in columns 4 7 Four columns are provided for allele designations to accommodate mixed samples The first four labeled peaks within each locus appear in the table The remaining five table columns are as follows Overflow If more than two peaks are labeled at one locus the text gt two labels will appear in this column Low Signal If the height of any peak at a locus is greater than the PAT specified in the GeneScan Analysis Parameters but less than 150 RFU the text lt 150 RFU will appear in this column Saturation If the raw data si
101. he AmpFZSTR Identifiler kit The AmpFZSTR Identifiler Allelic Ladder contains the majority of alleles reported for the 15 loci Protocol for 310 Genetic Analyzer with Mac OS Off Scale Data Overview f too much sample DNA is added to the PCR reaction mixtures the fluorescence intensity from the PCR products may exceed the linear dynamic range for detection by the instrument This is referred to as off scale data Multicomponent analysis cannot be performed accurately on data that is off scale Samples with off scale peaks will exhibit raised baselines and or excessive pull up of one or more colors under the off scale peaks Analyzed data from off scale peaks should not be used for quantitative comparisons For example the stutter peak that corresponds to an off scale main peak is likely to be overestimated Off Scale Data on To determine if data is off scale on the ABI PRISM9 310 Genetic the ABI PRISM 310 Analyzer Step Action 1 In the GeneScan Analysis Software highlight the sample file row for the questionable sample in the Analysis Control window Alternatively under View select Show Offscale Regions to highlight off scale data with a red bar The width of the red bar corresponds to the amount of data that is off scale Under the Sample menu choose Raw Data Examine the fluorescence intensity for the raw data peaks Any peaks that are greater than 8191 relative fluorescence units RFU are off
102. he allele designation and or desired text then click OK 5 6 Genotyping for the Macintosh OS Plot Window Viewing Options To zoom in and out on regions of the plot window Step Action 1 Click and drag in a region of an electropherogram in the Plot window to draw a box around the desired size range the vertical size of the box is not important Type 8 R hold down the command key and type the letter R to zoom in Type d H to zoom out completely To view electropherograms from more than one dye color in the Plot window Step Action 1 Under the Views menu choose Show Dye Lanes Window 2 Click on the desired Dye lane rows Note Hold down the Shift key on the keyboard to select multiple adjacent Dye lane rows Hold down the Command 85 key to select Dye lane rows that are not adjacent Under the Views menu choose Show Plot Window Making Tables Three macros for making tables are included in the AmpFZSTR Identifiler Kit Template They are a Make Allele Table b Make CODIS Table c 310 Make Table Two of these tables Make Allele Table and Make CODIS Table are very simple and contain only Sample Info and genotype data The other table 310 Make Table contains additional information The contents and unique features of each table are described below All four of the tables have two features in common A locus that has no labeled peaks will
103. he necessary size template for amplification Experiments and Results 4 37 E 125 150 ys 200 225 250 zs 200 325 350 1080 p o An Aada AA A m SB Ad 1 minute BE 50 A41 minute LI sr w d minute BE SR A4 1 minute 000 seo o BR 168 89 AMEL 7 AMEL 000 E o EIE 12v 82019019 020 seo BE 76 4503 03 1060 540 S NA GM 18 4108 08 10s se 3 AA BR 17R 8705 05 1080 se o DH 137 83 NA v WA 1096 se B j A E 86 As THON THOT 000 540 o A TRET 100 seo o BM eR eS FGA rGA 1080 se o AN GM 96 47 013 013 000 se o EHE 147 64 TPOX 7 TPOX 1096 se o n NH 67 1080 540 o GM 10690167016 100 540 o LH reves pie p18 1080 se o 6B A4 CSF CSF 1000 E o BE 110 8102 02 Figure 4 15 Multiplex and singleplex amplifications of a DNA sample incubated for 1 min with DNase I analyzed on the ABI Prism 310 Genetic Analyzer 4 38 Experiments and Results Mixture Studies 8 1 2 2 Mixture Studies Mixed Specimen Studies Species specificity sensitivity stability and mixture studies are conducted DAB 1998 Evidence samples may contain DNA from more than one individual The possibility of multiple contributors should be considered when interpreting the results We recommend that individual laboratories assign a minimum pe
104. her methods including phenol chloroform salting out and binding to silica particles Degraded DNA gives the same results as fully intact DNA over a wide range of average DNA sizes However DNA quantity can be underestimated when the DNA is extremely degraded For example experimental results indicated that the signal obtained for DNA degraded to an average size of 500 2000 bp was about half of the expected intensity Extremely degraded DNA usually amplifies less efficiently than intact DNA so a greater quantity of degraded DNA may be required to give the same results as intact DNA Commonly Asked Questions about the QuantiBlot Kit How Much DNA Multiple Film Exposures How much of the DNA extract should be added to the amplification reac tion if a sample gives no signal for the QuantiBlot assay As an example assume that 5 uL of the DNA extract is spotted and the lowest DNA standard tested is 150 pg So the DNA concentration in the sample must be less than 150 pg 5 uL or 30 pg uL The quantity of DNA in 10 uL of extract which is the maximum that can be added to an AmpFZSTR Identifiler kit amplification would therefore be less than 0 3 ng The possible approaches that can be taken for such a sample include the following Attempt amplification using 10 uL of the extract Concentrate the sample to a smaller volume using a Centricon 100 before amplification Is it possible to perform multiple film exposures with
105. ibed on the following page Genotyping for the Macintosh OS 5 19 Analyzing AmpF STR Identifiler Allelic Ladder To size the AmpFZSTR Identifiler Allelic Ladder alleles Step Action 1 Analyze the lanes injections containing allelic ladder using the same parameters used for samples Results To compare the lanes or injections of AmpFZSTR Identifiler Allelic Ladder Step Action 1 Compare the base pair sizes of one lane or injection of allelic ladder to those obtained for the other lanes or injections of allelic ladder All corresponding peaks peaks at the same position in the allelic ladder should be within 0 5 bp of each other 2 If one or more corresponding peaks are not within 0 5 bp of each other check the GeneScan 500 LIZ Size Standard peaks in all allelic ladder lanes or injections to confirm that all GeneScan 500 LIZ Size Standard peaks have been assigned the correct size and or that all peaks are clearly resolved Genotyping Samples Manually To manually genotype samples Step Action 1 Select one lane or injection of allelic ladder to use for genotyping Note Our studies have shown that it does not matter which lane or injection of allelic ladder is selected if the alleles in the allelic ladder samples are within 0 5 bp of each other 2 Compare the base pair size obtained for each sample allele peak to the sizes obtained for the allelic ladder peaks 3 As
106. icity and sensitivity 1 0 mM 1 15 mM m l Ada oon ah Ai ul m 2400 1 25 mM 1200 al Standard 0 ad la oon ad had N Concentration 2400 1 35 mM 1200 o Juin ada di d x TN h Ay 1 50 mM Figure 4 1 A 1 ng amplification of genomic DNA varying the magnesium chloride concentration analyzed on the ABI PRISM 310 Genetic Analyzer Experiments and Results 4 3 Thermal Cycler Thermal cycling parameters were established for amplification of the Parameters AmpFZSTR Identifiler kit in the GeneAmp PCR Systems 9600 and 9700 Thermal cycling times and temperatures of GeneAmp PCR Systems were verified Annealing and denaturation temperature windows were tested around each stipend to verify that a 1 5 C window produced a specific PCR product with the desired sensitivity of at least 1 ng of AmpFZSTR Control DNA 9947A The effects of denaturation and annealing temperatures on the amplification of AmpFZSTR Identifiler kit loci were examined using AmpF STR Control DNA 9947A and two DNA samples The denaturation temperatures tested were 92 5 94 and 95 5 C all for 1 minute hold times on the GeneAmp PCR System 9700 The annealing temperatures tested were 55 57 59 61 and 63 C see Figure 4 2 also for 1 minute hold times in the GeneAmp PCR System 9700 The PCR products were analyzed using the ABI PRISM 310 Genetic Analyzer Neither preferential nor differential amplification was observed in the dena
107. ility of Identity 4 56 Probability of Paternity Exclusion 4 57 Experiments and Results 4 1 Experiments Performed Using the AmpFZSTR Identifiler PCR Amplification Kit Importance of Validation of a DNA typing procedure for human identification Validation Experiments applications is an evaluation of the procedure s efficiency reliability and performance characteristics By challenging the procedure with samples commonly encountered in forensic and parentage laboratories the validation process uncovers attributes and limitations which are critical for sound data interpretation in casework Sparkes Kimpton Gilbard et al 1996 Sparkes Kimpton et al 1996 Wallin et al 1998 Experiments to evaluate the performance of AmpFZSTR Identifiler PCR Amplification Kit were performed at Applied Biosystems Some of these experiments were performed according to the DNA Advisory Board DAB Quality Assurance Standards effective October 1 1998 DNA Advisory Board 1998 The DNA Advisory Board issued quality assurance standards for forensic DNA testing laboratories These DAB standards describe the quality assurance requirements that a laboratory should follow to ensure the quality and integrity of the data and competency of the laboratory DAB defines a laboratory as a facility in which forensic DNA testing is performed Based on these standards Applied Biosystems has conducted experiments which comply with Standards 8 1 1 and 8
108. ince the presence of BSA can improve the amplification of DNA from blood containing samples BSA has been included in the AmpF STR PCR Reaction Mix at 4 ug per 25 uL amplification BSA has also been identified as an aid in overcoming inhibition from samples containing dyes such as in denim Comey et al 1994 To examine the effects of hematin on the amplification results obtained by the AmpF STR Identifiler kit DNA samples were amplified using the AmpFZSTR Identifiler kit reagents including the BSA containing PCR reaction mix in the presence of varying concentrations of purified hematin The concentrations of hematin used were 0 uM 10 uM 12 uM 14 uM 16 uM 18 uM and 20 uM When the amount of hematin was 4 34 Experiments and Results increased to a concentration that started to inhibit the PCR CSF1PO and D281338 were the first loci to exhibit decreased amplification followed by D16S539 and D18S51 Differential amplification was observed in the presence of increasing amounts of hematin Moreover as the concentration of hematin was increased the overall yield of products was reduced particularly for the larger loci 180 300 330 LLL id BENTTV A i ME 38 41 BW No hematin BW No hematin 36 A1 BW No hematin BW No hematin DR SY A1 BW No hematin BW No hematin 3R A1 BW No hematin BW No hematin 48003 32001 16003 i of a ada l an ar i1 i a wt a GM 48 43 BW 10uM
109. indow Protocol for 310 Genetic Analyzer with Mac OS 3 15 To create a five dye sample sheet continued Step Action 4 In the five dye Sample Sheet Enter sample name sample information and comments Designate color for appropriate size standard Save Be sure to select the orange dye as the designated size standard for all five dye samples Under Preferences this feature can be preset See page 3 11 Ei Sample Sheet untitled B GeneScan Sample Sheet Sample Info Comments Setting up five dye samples requires the use of a five dye sample sheet You may not set up both four dye and five dye samples in a five dye sample sheet All four dye samples must be set up separately in a four dye specific sample sheet To create a new injection list choose New from the File menu The Create new window appears Create new w mj Tu fo Tu Tu Sequence GeneScan Sequence Sequence GeneScan GeneScan Injection Injection Smpl Sheet Smpl Sheet Smpl Sheet Smpl Sheet Cancel List List 48 Tube 96 Tube 48 Tube 96 Tube Lej Choose the GeneScan Injection List icon 3 16 Protocol for 310 Genetic Analyzer with Mac OS To create a five dye sample sheet continued
110. ist and then choose Show Step Window from the Views menu The first filter step for this macro which applies to the sample alleles reads Remove labels from peaks whose height is less than 20 of the highest peak in a category s range Note that this particular option does not include any condition regarding the bp size of the filtered peak relative to a higher peak Indeed this second filtering option will remove labels from all peaks that are less than a specified percentage of the highest peak observed anywhere in the locus range To edit the filter value Step Action 1 Click on this step in the Step window 2 Choose Edit Step from the Macro menu Note This macro uses the second filter option in the Filter Labels window 3 If desired change the value from 20 to some other value 4 Click Replace The Kazam 20 filter macro is provided as an option for laboratories that would like to use one general filter value for all loci This macro can also be used when a high level of filtering specificity is not required as in the typing of single source samples e g database samples The original AmpFZSTR Identifiler Template File can be modified so that the changes made to the macros or settings are used as the default To modify the template Step Action 1 Close all Genotyper windows but do not quit the application Genotyping for the Macintosh OS 5 15 To modify the
111. ition The sample can be re amplified using less DNA When the total number of allele copies added to the PCR is extremely low unbalanced amplification of the two alleles of a heterozygous individual may occur Walsh et a 1992 Wallin et a 1998 due to stochastic fluctuation in the ratio of the two different alleles Sensabaugh et al 1991 The PCR cycle number and amplification conditions have been specified to produce low peak heights for a sample containing 20 pg human genomic DNA Low peak heights should be interpreted with caution Individual laboratories may find it useful to determine an appropriate minimum peak height threshold based on their own results and instruments using low amounts of input DNA 4 32 Experiments and Results 0 125ng ow vee Gee 0 062 5ng cR d e 0 03 1ng 0 01 6ng Negative Control Figure 4 12 Effect of amplifying various amounts of DNA ranging from 16 pg to 1 ng Note that the y axis scale is magnified for the lower amounts of DNA analyzed using the ABI PRISM 310 Genetic Analyzer Experiments and Results 4 33 Stability 8 1 2 2 Stability Overview Differential and Preferential Amplification Effect of Inhibitors Species specificity sensitivity stability and mixture studies are conducted DAB 1998 Lack of Amplification of Some Loci As with any multi locus system the possibilit
112. itz E 1969 Eine neue farbemethode zum nachweis der spermein bei sittlichkeitsdelicten Arch Kriminol 144 145 148 Prince A M and Andrus L 1992 PCR How to kill unwanted DNA Biotechniques 12 358 Puers C Hammond H Jin L Caskey C and Schumm J 1993 Identification of repeat sequence heterogeneity at the polymorphic short tandem repeat locus HUMTHO 1 AATG and reassignment of alleles in population analysis using a locus specific allelic ladder Am J Hum Genet 53 953 958 References F 7 F 8 References Sambrook J Fritsch E F and Maniatis T eds 1989 Molecular Cloning A Laboratory Manual 2nd Edition Volume 2 Cold Spring Harbor Laboratory Press New York pp E10 E14 Sensabaugh G F 1982 Biochemical markers of individuality In Saferstein R ed Forensic Science Handbook Prentice Hall Inc New York pp 338 415 Sensabaugh G F von Beroldingen C The polymerase chain reaction application to the analysis of biological evidence In Farley MA Harrington JJ editors Forensic DNA Technology Michigan Lewis 1991 63 82 Sharma V and Litt M 1992 Tetranucleotide repeat polymorphism at the D21S11 locus Hum Mol Genet 1 67 Singer Sam J and Tanguay R 1989 Use of Chelex to improve the PCR signal from a small number of cells Amplifications 3 11 Smith R N 1995 Accurate size comparison of short tandem repeat alleles amplified by PCR Biotechniques 18 122 128 Spark
113. ive a new MSDS packaged with a hazardous chemical PN ablic CHEMICAL HAZARD Be sure to familiarize yourself with the MSDSs before using reagents or solvents Introduction 1 9 Ordering MSDSs You can order free additional copies of MSDSs for chemicals manufactured or distributed by Applied Biosystems using the contact information below To order MSDSs Then Over the Internet a Go to our Web site at www appliedbiosystems com techsupp b Click MSDSs If you have Then The MSDS document number or the Document on Demand index number Enter one of these numbers in the appropriate field on this page The product part number Keyword s Select Click Here then enter the part number or keyword s in the field on this page C You can open and download a PDF using Adobe Acrobat Reader of the document by selecting it or you can choose to have the document sent to you by fax or email By automated telephone service Use To Obtain Documents on Demand on page A 6 By telephone in the United States Dial 1 800 327 3002 then press 1 By telephone from F Canada To order in Dial 1 800 668 6913 and English Press 1 then 2 then 1 again French Press 2 then 2 then 1 By telephone from any other country See To Contact Technical Support by Telephone or Fax on page A 2 For chemicals not m
114. l DNA for typing J Forensic Sci 45 3 674 676 Technical Working Group on DNA Analysis Methods 1995 Guidelines for a quality assurance program for DNA analysis Crime Lab Digest 22 21 43 Urquhart A Oldroyd N J Kimpton C P and Gill P 1995 Highly discriminating heptaplex short tandem repeat PCR system for forensic identification Biotechniques 18 116 121 U S Department of Health and Human Services 1993 Biosafety in Microbiological and Biomedical Laboratories 3rd edition U S Government Printing Office U S Department of Health and Human Services OSHA Bloodborne Pathogen Standard 29 CFR part 1910 1030 Wallin J Buoncristiani M Lazaruk K Fildes N Holt C and Walsh P 1998 TWGDAM validation of the AmpFISTR Blue PCR Amplification Kit for forensic casework analysis J Forensic Sci 43 4 854 870 Wallin J et al 2001 Constructing universal multiplex PCR systems for comparative genotyping Journal of Forensic Sciences in press Walsh P S Metzger D A and Higuchi R 1991 Chelex 100 asa medium for simple extraction of DNA for PCR based typing from forensic material Biotechniques 10 506 518 Walsh P S Erlich H A and Higuchi R 1992 Preferential PCR amplification of alleles mechanisms and solutions PCR Methods Appl 1 241 250 Walsh P S Varlaro J and Reynolds R 1992 A rapid chemiluminescent method for quantitation of human DNA Nucleic Acids Hes 20 5061 50
115. l Down from the Edit menu IMPORTANT The matrix file must be one that was made using the 6 FAM VIC NED PET and LIZ matrix standards and Filter Set G5 module Furthermore if you wish to autoanalyze a copy of the matrix file must be placed in the ABI folder located in the System Folder Click the Run button Note If you have not preheated the heat plate the module has an initial step in which the plate is heated to 60 C before running the first sample This step takes up to 30 min Once the plate reaches 60 C the run will begin Protocol for 310 Genetic Analyzer with Mac OS 3 25 Setting Up Software Parameters Setting the Perform the following steps in GeneScan Software v3 1 or higher To set the analysis parameters Action Launch the GeneScan Software v 3 1 From the Settings menu select Analysis Parameters Note A more detailed discussion can be found for each of the six Analysis Parameters in the GeneScan Analysis 3 1 User s Manual Analysis Parameters Step 1 2 3 Fill in the dialog box a Analysis Range Choose This Range Data Points radio button Enter Start and Stop data point numbers in the entry fields The Start data point should be a selected data point just before the first peak of interest the 75 bp size standard peak At a minimum the Stop data point should be a selected data point just after the last peak of interest the 450 bp size standard
116. le peaks in the allelic ladder and to determine the correct offset values for each allele category Finding and recognizing the Leftmost first Allele Peak in Each Allelic Ladder Identification of the leftmost peak is accomplished through the specifications of the first os category listed within each group of offset categories This first os category 12 0s in the case of D381358 is specified to find all peaks in a range of 7 bp around the reference size for the indicated allele Each Calculate locus Offsets macro applies a percentage filter to all peaks in the 7 bp range in the allelic ladder This avoids the first stutter peak in each allelic ladder and thus identifies the first allele peak as the leftmost peak Calculating the Offset Values Categories with the os suffix are called offset categories and are described below The base pair size indicated in each category is a reference size One main function of the macros in the AmpFZSTR Identifiler Template is to offset the reference sizes relative to the sizes obtained for the alleles in the allelic ladder These offset steps are performed by the Calculate locus Offsets macros located in the Macro list of the Genotyper software Main window After the macros are run the calculated offset values are indicated in parentheses near the end of each category line in the Categories window An example of how to interpret the offset values is given here for D3S1358
117. lification DNA extracted with Chelex cannot be used for RFLP analysis D 2 DNA Extraction Protocols FTA Paper Extraction The FTA paper extraction begins immediately when blood is spotted on FTA paper The cells are lysed and the DNA is immobilized within the matrix of the paper The DNA is purified by performing a series of washes after which the DNA is ready for PCR amplification Warnings to Users Read the Material Safety Data Sheet MSDS and label warning furnished by the supplier of each chemical or reagent for the correct handling and the proper use of protective equipment For additional copies of an MSDS call 1 800 327 3002 Collection and Storage of Samples for DNA Extraction Proper Collection Storage of various DNA specimens is an essential step to insuring that the DNA profiles obtained are accurate and meaningful Proper chain of custody is vital to maintaining the integrity of each particular specimen DNA Extraction Protocols D 3 DNA Quantitation Overview About This This appendix discusses the importance of quantitating DNA samples Appendix prior to amplification The QuantiBlot Human DNA Quantitation Kit is described and can be used for the quantitation of samples In This Appendix Appendix E contains the following topics Topic See Page Overview E 1 Importance of Quantitation E 2 Using the QuantiBlot Kit E 3 Commonly Asked Questions about the QuantiBlot Kit E 5 DNA Quantitatio
118. llow the handling instructions Wear appropriate protective eye wear clothing and gloves Be sure to include at least one injection of AmpFZSTRS9 Identifiler Allelic Ladder per run in the calculations 2 a Vortex the tube to mix b Spin the tube briefly in a microcentrifuge 3 a Label tubes as appropriate b Aliquot 25 uL of Hi Di Formamide GeneScan 500 LIZ solution into 0 2 mL or 0 5 mL Genetic Analyzer sample tubes Note To pipet the Hi Di Formamide size standard solution we recommend using a repeating pipettor 4 Add 1 5 uL of PCR product or AmpFZSTR Identifiler Allelic Ladder per tube Mix by pipetting up and down 5 Seal each tube with a septum 6 Vortex the sample tray and spin briefly in a microcentrifuge Note Ensure that there are no bubbles 7 Denature each sample for 3 min at 95 C 8 Chill tubes for at least 3 min on ice Note Be careful not to carry over any water on the outside of the tubes Water on the autosampler tray may promote arcing 3 22 Protocol for 310 Genetic Analyzer with Mac OS Loading Samples To load samples Step Action 1 Open the instrument door and press the Tray button to present the autosampler 2 Place a 48 well or 96 well sample tray on the autosampler For a 48 well autosampler tray tube 1 will go into sample tray position A1 tube 2 into sample tray position A3 etc For a 96 well autosampler tray tube 1 will go into sa
119. locus in PET dye Experiments and Results 4 31 Sensitivity 8 1 2 2 Sensitivity Species specificity sensitivity stability and mixture studies are conducted DAB 1998 Effect of DNA Quantity on Results Importance of The amount of input DNA added to the AmpFZSTR Identifiler PCR Quantitation Amplification kit should be between 0 5 and 1 25 ng The DNA sample should be quantitated prior to amplification using a system such as the QuantiBlot Human DNA Quantitation Kit P N N808 0114 see Appendix E The final DNA concentration should be in the range of 0 05 0 125 ng uL so that 0 5 1 25 ng of DNA will be added to the PCR reaction in a volume of 10 uL If the sample contains degraded DNA amplification of additional DNA may be beneficial If too much DNA is added to the PCR reaction then the increased amount of PCR product that is generated can result in the following Fluorescence intensity that exceeds the linear dynamic range for detection by the instrument off scale data Off scale data is a problem for two reasons Quantitation peak height and area for off scale peaks is not accurate For example an allele peak that is off scale can cause the corresponding stutter peak to appear higher in relative intensity thus increasing the calculated percent stutter Multicomponent analysis of off scale data is not accurate which results in poor spectral separation pull up Incomplete A nucleotide add
120. ls should be limited through careful handling of sample tubes and reagents Applied Biosystems does not intend these references for laboratory design to constitute all precautions and care necessary using PCR technology Extra precautions and care should be taken during DNA extraction and PCR setup to prevent transfer of DNA from one sample to another Use a new filter plugged pipet tip for each sample open tubes carefully and keep sample tubes closed when not in use Applied Biosystems does not intend these references for laboratory design to constitute all precautions and care necessary when using PCR technology DNA Extraction Protocols Overview About This Appendix D describes some extraction methods for various DNA Appendix samples In This Appendix Appendix D contains the following topics Topic See Page Overview D 1 Introduction D 2 Collection and Storage of Samples for DNA Extraction D 3 DNA Extraction Protocols D 1 Introduction Overview of DNA Sample Types DNA Extraction Methods Many DNA extraction procedures have been developed Both manual and automated extraction procedures can be further divided into organic and non organic procedures Depending upon the material received the scientist will need to determine which procedure is appropriate for each piece of evidence DNA for PCR amplification and analysis using the AmpF STR Identifiler PCR Amplilfication Kit may be extracted
121. lter Set G5 module files must be installed on the instrument s computer before making a matrix file using the 6 FAM VIC NED PET and LIZ matrix standards Filter Set G5 module files must also be used on all subsequent runs Samples that are run on a capillary using Filter Set G5 must be analyzed using a matrix file that was created using Filter Set G5 3 14 Protocol for 310 Genetic Analyzer with Mac OS Five Dye Data Collection Overview Creating a Five Dye Sample Sheet and Injection List The ABI PRiSM 310 Data Collection Software v2 1 enables collection of five dye data for DNA fragment analysis applications This section provides detailed information on sample sheet and injection lists To create a five dye sample sheet Step Action 1 From the File menu choose New The Create new window appears Create new Sequence GeneScan Sequence Sequence GeneScan GeneScan Injection Injection Smpl Sheet Smpl Sheet Smpl Sheet Smpl Sheet Cancel List List 48 Tube 96 Tube 48 Tube 96 Tube L_cencel 2 Choose the icon corresponding to an appropriate GeneScan Sample Sheet configuration A Sample Sheet window appears Ez Sample Sheet untitled EB GeneScan Sample Sheet 3 Choose the 5 Dyes option from the drop down menu in the upper right corner of the w
122. lting from incomplete A nucleotide addition due to omission of the 60 minute extension step The AmpliTaq Gold DNA Polymerase generally requires extra time to complete the A nucleotide addition at the 3 end of the PCR products Lack of full A nucleotide addition may be observed in AmpFZSTR Identifiler kit results when the amount of input DNA is greater than recommended protocols The reason for this is that more time is needed for AmpliTaq Gold DNA Polymerase to add the A nucleotide to all molecules as more PCR product is generated Amplification of too much input DNA will also result in off scale data Artifacts Artifacts or anomalies have been seen in data produced on the ABI Prism 310 Genetic Analyzer when using the AmpFZSTR Identifiler kit The shape of these artifacts is not consistent with the shape of labeled DNA fragments as seen on the ABI PRISM 310 Genetic Analyzer Artifacts may or not be reproducible Figure 4 10 on page 4 27 demonstrates reproducible artifacts while using the AmpFZSTR Identifiler kit A user of the AmpFZSTR Identifiler kit on the ABI PRISM 310 Genetic Analyzer should consider these artifacts when interpreting data 4 26 Experiments and Results s 100 125 150 1 20 3 Orman aed Teraa Figure 4 10 Reproducible anomalies in the blue green yellow and red dye electropherograms when using the AmpFZSTR Identifiler PCR Amplification Kit Genotyping may result in the detec
123. lumn by clicking in the Sample File title row b Under the Sample menu select Install New Matrix c Choose the new matrix file located in the ABI folder within the System folder and click Open Analyze the matrix standard samples as follows a Under the Settings menu select Analysis Parameters and verify that the settings are correct b In the Analysis Control window select all five colors in each sample row for all of the matrix standard samples c Click the Analyze button 3600 4200 4800 5400 000 3200 2400 1600 800 TETE 3200 2400 1600 s00 LO U Lr 3200 2400 1600 s00 LL al a i 3200 2400 1600 800 E ELE a an T 3200 2400 1600 800 0 HL d uu po Figure 3 5 This figure exhibits the analyzed data of each matrix standard analyzed on the ABI PRISM 310 Genetic Analyzer 3 20 Protocol for 310 Genetic Analyzer with Mac OS To verify the accuracy of the matrix file continued Step Action 3 a In the Results Control window examine the results for all five colors for each of the matrix standard samples For example the 6 FAM matrix standard results should have peaks for Blue Evaluate the baseline A pattern of pronounced peaks or dips in any of the other four colors indicates that the color separation may not be optimal Examine the results for e
124. mple tray position A1 tube 2 into sample tray position A2 etc 3 Press the Tray button on the instrument to retract the autosampler Close the instrument door Protocol for 310 Genetic Analyzer with Mac OS 3 23 Sample To run the samples Electrophoresis Step Action 1 If not already open launch the ABI PRISM 310 Data Collection Software v2 1 From the File menu select New and click the appropriate GeneScan Smpl Sheet icon Note The 310 Genetic Analyzer Data Collection Software v 2 1 must be installed for use with the AmpFZSTR Identifiler PCR Amplification Kit Complete the sample sheet The sample sheet can be prepared at any time before the preparation of samples and saved in the Sample Sheet folder a Select 5 dyes from the drop down menu b Enter sample names numbers for each injection in the Sample Name column This will later indicate which sample is in which tube of the sample tray c Enter the sample description for each row in the Sample Info column for Blue Green Yellow and Red for each sample This is necessary for the AmpFZSTR Identifiler Template File to build tables containing the genotypes for each sample Type the word Ladder for the Blue Green Yellow and Red rows for the AmpFZSTR Identifiler Allelic Ladder injection Note Software requires the word Ladder See page 5 10 Note Alternatively a Select 5 dyes from the drop down menu b Ent
125. mples 4 40 to 4 41 guidelines laboratory setup C 1 validation studies 4 2 to 4 36 H help See technical support A 2 hematin effect on DNA samples 4 33 to 4 34 Index 2 I Internet address customer training information A 6 Documents on Demand A 6 L laboratory setup C 1 loci allele frequencies in the population databases 4 44 to 4 53 differential amplification 4 34 lack of amplification effect of DNA quantity on results 4 31 to 4 32 population data 4 43 to 4 53 allele frequencies 4 44 to 4 53 probability of identity 4 55 probability of paternity exclusion 4 56 samples used in studies 4 43 locus See loci M matrix file 3 12 P PCR amplification of tetranucleotide STR loci stutter peak 4 18 to 4 23 inhibitor causing lack of amplification 4 33 to 4 34 minimizing E 2 troubleshooting B 1 to B 5 PCR product precautions when handling the PCR product C 2 using too much PCR product E 2 See Also PCR phenol chloroform DNA extraction D 2 population genetics 4 43 to 4 52 allele frequencies 4 44 to 4 53 populations and samples used in the studies 4 43 Probability of Exclusion Pg from Paternity 4 56 probability of identity 4 55 Probability of Identity P 4 55 probability of paternity exclusion 4 56 See Also allele protocols ABI PRISM 310 Genetic Analyzer 3 3 to 3 37 data analysis 3 26 to 3 29 ending the run 3 34 preparing and loading samples 3 22 to 3 23 sample electrophoresis 3 24 to 3 25 setting up a run 3
126. n E 1 Importance of Quantitation DNA Quality Quantitation and PCR Amplification E 2 DNA Quantitation The DNA quality degree of degradation purity and total quantity in a sample influences the efficiency of a PCR amplification Lack of amplification is usually due to highly degraded DNA the presence of PCR inhibitors insufficient DNA quantity or any combination of these factors The QuantiBlot Human DNA Quantitation Kit P N N808 0114 is an ideal method for accurate quantitation of human DNA Walsh et al 1992 If the QuantiBlot kit determines that sufficient DNA is present in the extracted sample greater than approximately 0 05 ng uL concentration then lack of amplification is most likely due to PCR inhibitors or severe degradation of the DNA Quantitation of samples shows if there is a sufficient amount of DNA present for amplification Also PCR inhibition can be minimized by adding the smallest volume of DNA extract necessary for successful amplification volume containing approximately 0 5 1 25 ng Lastly by using the minimal volume of extracted DNA for PCR the number of different genetic marker tests or repeat analyses that can be performed is maximized Likewise informed decision s can be made regarding typing of samples present in extremely limiting quantities DNA quantitation is particularly important for amplifications using the AmpFZSTR Identifiler kit where optimal results are obtained using a r
127. neScan 500 LIZ mixture by pipetting up and down several times Loss of resolution after 100 bp Excess salt in sample Do not concentrate PCR product by evaporation Use Centricon 100 if necessary Too much DNA in sample Treat and dilute the PCR product Bad water Incorrectly prepared and or old solutions Use autoclaved or freshly prepared deionized water Replace buffer and polymer with fresh solutions Runs get progressively slower i e size standard peaks come off at higher and higher scan numbers Leaking syringe polymer is not filling capillary before every injection Clean syringe thoroughly Replace syringe Runs get progressively faster i e size standard peaks come off at lower and lower scan numbers Water in syringe Prime syringe with small volume of polymer and discard Fill syringe with polymer High baseline Dirty capillary window Clean capillary window with 9596 ethanol Capillary moved out of position in laser window Position capillary in front of laser window Cracked capillary Replace the capillary Troubleshooting B 5 Laboratory Setup Overview About This This appendix provides some references for laboratories preparing to Appendix implement PCR technology Careful planning and design of the laboratory and training of all laboratory personnel are necessary to ensure that exogenous DNA and PCR products are confined
128. ns Sizes may differ between protocols for the same instrument platform because of differences in gel or polymer concentration run temperature gel or capillary thickness and well to read length Slight procedural and reagent variations between gels or between single and multiple capillaries result in greater size variation than that found between samples on the same gel or between samples injected in the same capillary in a single run Size Standard The GeneScan 500 LIZ Size Standard should be used with the AmpF4ZSTR Identifiler kit Common alleles for all AmpFZSTR Identifiler kit loci are less than 400 base pairs The recommended sizing method Local Southern utilizes two internal lane size standard peaks larger than each allele and two smaller than each allele to be sized Thus when size standard peaks are defined in routine analyses inclusion of the 400 base pair and 450 base pair peaks in the GeneScan 500 LIZ Size Standard is recommended The internal lane size standard is run with every sample AmpF STR Identifiler kit PCR products and AmpFZSTR Identifiler Allelic Ladder and is used to normalize lane to lane or injection to injection migration differences thereby providing excellent sizing precision within a gel or within a set of capillary injections Size windows based on the allelic ladder are used to assign allele designations to the samples The procedure for running the allelic ladder and determining genotypes is descr
129. nsion Memorial Blood Center of Minneapolis and the Federal Bureau of Investigation Native American 191 samples were provided by the Minnesota Bureau of Criminal Apprehension Memorial Blood Center of Minneapolis 4 44 Experiments and Results AmpF STR Table 4 3 shows the AmpF STR Identifiler kit allele frequencies in four Identifiler Kit populations listed as percentages Allele Frequencies Table 4 3 AmpFZSTR Identifiler kit allele frequencies African American U S Caucasian U S Hispanic Native American Allele n 357 n 349 n 290 n 191 CSF1PO 6 7 4 62 0 14 0 34 9 8 7 56 0 29 0 17 0 52 9 3 78 1 72 0 86 8 38 10 27 87 24 21 23 10 30 89 11 20 59 31 81 28 28 21 99 11 3 0 14 E 12 29 13 32 81 39 66 32 72 13 5 32 7 31 6 38 4 71 14 0 98 1 43 0 86 0 79 15 A 0 29 0 34 K D2S1338 15 0 14 2 S 16 5 32 4 73 2 41 2 62 17 10 78 17 34 21 21 9 95 18 5 60 6 30 4 14 7 07 19 14 15 13 75 22 76 29 58 20 6 02 14 61 13 79 9 69 21 14 01 2 58 2 59 2 36 22 13 17 4 01 7 41 15 18 23 10 78 11 46 11 38 11 78 24 9 80 11 75 8 45 7 85 25 8 12 10 60 5 17 3 14 26 1 96 2 72 0 69 0 79 27 0 14 0 14 5 2g Experiments and Results 4 45 Table 4 3 AmpFZSTR Identifiler kit allele frequencies continued African American U S Caucasian U S Hispanic Native American Allele n 2 357 n 2 349 n 290 n 191 D3S1358 lt 11 0 42 0 14 N g 11 9 E T 0 26 12 0 56 i 0 17 13 0 70 0
130. omment on page 4 17 regarding stutter at allele 15 of D381358 4 22 Experiments and Results 18 1 17 4 16 4 15 4 D 14 4 M i T 12 4 H 3 N B H T N i 11 4 R ka oe N d e gen 8 x 10 4 PM e 3 e e 38 E t o UEZ Z 39 iit 5 Y a E gerade e se 5 2384 1 93 e oe 5 oo 9 3709 1 U 741 of tgs a E LY TZ oe E L 6 HU E e tp 333 5 4 Sas 2 33 4 8 T R H 3 ie L 1 38 24 D 1 d 0 toot toto 9 10 11 12 13 14 15 16 17 11 12 13 14 15 16 17 18 19 20 21 6 7 8 9 10 11 12 11 12 13 14 15 16 17 18 19 20 21 22 23 D19S433 vWA TPOX D18S51 Allele Figure 4 6 Stutter percentages for the D19S433 vWA TPOX and D18S51 loci Experiments and Results 4 23 16 4 14 4 U 12 4 T k 10 4 1 e E U 2 84 egese 5 3 t e t t t 7 8 te 3 S 6 k i a 1 3 H e N i i 44 24 I TTL toto ot 7 8 9 10 11 12 13 14 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 D5S818 FGA Allele Figure 4 7 Stutter percentages for the D58818 and FGA loci 4 24 Experiments and Results 1600 1200 800 Sample 1 400 0 160 120 Sample 1 80 40 0 150 C 120 Negative 80 Control 40 Figure 4 8 Sample 1 in panel A and panel B has a profile of 15 16 for D381358 The amount of stutter can not be accurately measure
131. omponent Analysis Overview About Multicomponent Analysis How Multicomponent Analysis Works Multicomponent analysis is the process that separates the five different fluorescent dye colors into distinct spectral components The four dyes used in the AmpFZSTRS Identifiler PCR Amplification Kit to label samples are 6 FAM VIC NED and PET dyes The fifth dye LIZ is used to label the GeneScan 500 Size Standard Each of these fluorescent dyes emits its maximum fluorescence at a different wavelength During data collection on the ABI PRISM O instruments the fluorescent signals are separated by a diffraction grating according to their wavelengths and projected onto a charge coupled device CCD camera in a predictably spaced pattern 6 FAM dye emits at the shortest wavelength and is displayed as blue followed by the VIC dye green NED dye yellow PET dye red and LIZ dye orange Although each of these dyes emits its maximum fluorescence at a different wavelength there is some overlap in the emission spectra between the dyes Figure 1 1 The goal of multicomponent analysis is to effectively correct for spectral overlap 6 FAM VIC NED PET LIZ A Q o 80 60 40 20 Normalized Emission o 500 550 600 650 700 Wavelength nm Figure 1 1 Emission spectra of the five dyes used in the AmpFZSTR Identifiler PCR Amplification Kit Introduction 1 3 Loci Amplified by The table below shows
132. on 1 In the Step Window for the Kazam macro scroll down to the line that reads Select category D3S1358 2 Five rows below click on the line that reads Remove labels from peaks followed by a 835 higher labeled peak within 3 25 to 4 75 bp Genotyping for the Macintosh OS 5 13 To filter stutter peaks Step Action 3 From the Macro menu choose Edit Step In the Filter Labels window that appears there are four options check boxes for filtering In this example the filtering option for D381358 is denoted in the last check box This filtering option includes another check box that reads higher by at least 835 For each labeled peak e g peak A in the locus size range this filtering option examines the very next e greater in bp size labeled peak peak B The label will be removed from peak A if peak B meets both of the specified criteria a peak B is higher by at least 835 b peak B is within 3 25 to 4 75 bp The percentage value in this filtering option is calculated as follows peak B peak A peak A x 100 percentage value For example if peak A 175 RFU and peak B 2500 RFU then the percentage value is calculated as follows 2500 175 175 x 100 1329 In this example the label will be removed from peak A provided that the filter option specifies a threshold of 835 and that peak B is within 3 25 to 4 75 bp of peak A Conventionally percent stutter
133. othing and gloves IMPORTANT Do not include the GeneScan 500 LIZ Size Standard in the preparation of the matrix standards 2 a Denature the samples at 95 C for 3 min b Quick chill on ice for 3 min c Place tubes in the appropriate sample tray Note Be careful not to carry over any water on the outside of the tubes Water on the autosampler tray may promote arcing 3 Launch the ABI PRISM 310 Collection application Under the File menu select New and click the GeneScan Smpl Sheet 48 Tube or GeneScan Smpl Sheet 96 Tube icon as appropriate 5 a Complete the sample sheet as described in the AB PRISM 310 Genetic Analyzer User s Manual b Enter the sample names numbers for each row in the Sample Name column This will identify which sample is in which tube of the sample tray c Close and Save the sample sheet 6 Under the File menu select New and click the GeneScan Injection List icon 3 18 Protocol for 310 Genetic Analyzer with Mac OS To make the matrix file continued Step Action 7 a In the Injection List select the appropriate sample sheet from the Sample Sheet pop up menu b From the Module pop up menu choose GS STR POP4 1 mL G5 for every injection c Choose None in the Matrix File column for each matrix standard sample Note Review data of each matrix standard Re inject if necessary 8 Click Run 2800 4200 4200 5400 8000 s IDA d do Lal hal
134. ple To edit the cells of the table a Click in a cell of the table that contains an allele designation b From the Edit menu select Edit Cell c Type the desired information in the box and click OK Print the table by selecting Print from the File menu Optional From the Table menu select Export to File to save the table as a Microsoft Excel readable document From the File menu select Save to save the template file with data Genotyping for the Macintosh OS 5 9 Understanding the AmpF STR Identifiler Kit Template Troubleshooting Automated To Troubleshoot Automated Genotyping Genotyping Observation Probable Cause Recommended Action Warning message Could not complete The word ladder is not in Sample Info for Type the word Ladder in Sample Info The word Ladder must be entered Run Macro the lane or command injection of allelic tor each dye color Blue Green Yellow and Red in Sample Info for because no ladder the AmpF STR Identifiler Allelic dye lanes are Lad pea le selected Pe Warning One or more Use another allelic ladder in the message Could peaks in the project or not complete allelic ladder are a Inthe GeneScan Analysis Run Macro below the Peak Software lower the Peak command Amplitude Amplitude Threshold values because the Threshold that for Blue Green Yellow and labeled peak was specified in Red dye colors in the
135. r oy A OJN Remove the tubes from the instrument block after the PCR is completed 6 Store the amplified DNA If you are storing the DNA Then place at lt 2 weeks 2 to 6 C gt 2 weeks 15 to 25 C IMPORTANT Protect the amplified products from light PCR Amplification 2 7 Amplification FTA treated DNA collection cards can be useful for the collection Using storage and processing of biological samples A small punch of the Bloodstained bloodstained card can be placed directly into an amplification tube FTA Cards Purified and amplified without transferring the evidence Our studies have indicated that a 1 2 mm bloodstained punch contains approximately 5 20 ng DNA Accordingly an appropriate cycle number for this high quantity of DNA is 25 cycles It is recommended that each laboratory determine the cycle number based upon individual validation studies In the example shown in Figure 2 1 a 1 2 mm punch of a bloodstained FTA card was purified using one wash with FTA Purification Reagent and two washes with 1X TE buffer After drying at room temperature overnight the punch was then amplified directly in the MicroAmp tube for 25 cycles E 120 150 180 210 240 270 300 330 2700 2400 2100 1800 1500 1200 a 4I AR 5 3B B2 FTA 25 cycles GM 36 B2rFTA25cycles LIE 3v B2rFTA25cycles BE 25 62 FTA 25 cycles Figure 2 1 AmpF
136. r 3 22 Loading Samples 2 0 0 0 cece eee eh 3 23 Sample Electrophoresis 00 00 cece eee eee 3 24 Setting Up Software Parameters 00000020 e eee eee 3 26 Setting the Analysis Parameters esses 3 26 GeneScan Software Results 0 0 0 0 eese 3 30 GeneScan Analysis Software 00 0 0 002 cee ee eee 3 30 Information Provided in the Electropherogram and Table 3 30 Standards for Samples 00 0 0 e eee eee eee ee 3 32 Otf ScaleDat zc 4e kent onde ie EE EU RC S Lee ee as 3 33 OVERVIEW oce Sb Sass made eor eR S ere os 3 33 Off Scale Data on the ABI PRISM 310 3 33 Shutting Down the Instrument eee 3 34 Ending the Run ue cR REIR IE VIAGRA 3 34 Dedicated Equipment and Supplies esee 3 35 Equipment Required 0 0 0 0 cece eee eee ee eee 3 35 Reagents Required 0 0 0 0 cee ee ee 3 36 Software and User Documentation Required 3 37 4 Experiments and Results OV rVIeW 2 4477 T 3 eho t ete cere SERM earth RN te E dane bey d About This Chapter In This Chapter 3 50 9 35 p44 RR RES pH er ee Rast Experiments Performed Using the AmpF STR Identifiler PCR Amplification Kit a 5 ipao T SE e R RT eee eee Importance of Validation 0 cee cece eee eee Experiment asco cto eee oda ews ate ew doen SR dat T N Developmental Validation 0 0 ccc cece ee eee eee 8 1 1 Developmental Validation 00 00008 PCR Components 15 06
137. r detection and quantitation down to 150 pg The chemiluminescent method can detect 150 pg with a 15 minute exposure to film and can detect as little as 20 pg with longer film exposures 3 hours to overnight Results obtained from various biological samples using the QuantiBlot Kit are shown in Figure E 1 on page E 4 Note For specific procedures refer to the QuantiBlot Human DNA Quantitation Kit product insert One significant advantage offered by the QuantiBlot kit is that the probe is highly specific for human primate DNA When tested 300 ng quantities of several non primate DNA samples E coli yeast dog cat mouse rat pig cow chicken fish and turkey were found to give either no signals or signals that were less than or equal to that obtained for 0 15 ng of human DNA This high degree of specificity for human primate DNA allows for the accurate quantitation of target human DNA in samples that also contain significant amounts of microbial or other non primate DNA DNA Quantitation E 3 Single Stranded and Degraded DNA E 4 DNA Quantitation 10 ng 10 ng Gum e p 5 p 5 2 5 Ce pd 2 5 0 3 0 3 0 15 0 15 Figure E 1 QuantiBlot Human DNA Quantitation Kit results ECL detection Another advantage of the QuantiBlot kit method is that single stranded and or non purified DNA samples can be quantitated DNA samples extracted using the Chelex method can be quantitated as can those extracted by ot
138. r resolution or high baseline Reinstall the pump block on the instrument after cleaning Installing the Syringe on the Pump Block To install the syringe on the pump block Step Action 1 Move the syringe drive toggle on the instrument to the left in order to be able to attach the syringe to the pump block Protocol for 310 Genetic Analyzer with Mac OS 3 7 To install the syringe on the pump block continued Step Action 2 Place the 1 0 mL syringe through the right hand port of the plastic syringe guide plate and screw the syringe into the pump block The syringe should be finger tight in the block Hand tighten the valves on the pump block to the left of and below the syringe Note Overtightening can cause microscopic fractures in the pump block Undertightening may result in syringe leak detected message Installing the Capillary To install the capillary Step Action 1 a Clean capillary window with 95 ethanol on a lint free tissue b Do not touch capillary window after cleaning a Install the 47 cm 50 um i d capillary P N 402839 green mark as described in the AB PRISM 310 Genetic Analyzer User s Manual b Follow the instructions in the section titled Installing the Capillary c If a new capillary has been installed select Change Capillary under the Instrument menu d Select OK in the Reset window to set the injection counter to ze
139. re application and open the template file simultaneously Note The AmpF4STR Identifiler Kit Template is a Stationery pad which means that a new document is created when the template file is opened The original template file is not overwritten Set preferences to import raw data Blue Green Yellow Red and Orange To import the GeneScan sample files a Under the File menu choose Import GeneScan File s b Select the project file and click Import If each sample does not already have Sample Info completed in the sample sheet this can be accomplished in Genotyper software as follows a Under the Views menu choose Show Dye lanes window b Select the first sample row by clicking on the row c Click the mouse cursor in the Sample Info box at the top of the window and type the sample designation or description d Repeat steps b and c to enter a sample description for every dye lane in the list Enter the same sample description for all dye colors of a single sample From the Macro list at the bottom left of the Main window select Check GS500 Under the Macro menu choose Run Macro In the plot window that appears scroll through each sample to verify that each GeneScan 500 peak from 75 450 bp was assigned the correct size in the GeneScan Analysis Software From the Macro list at the bottom left of the Main window select Kazam Under the Macro menu choose Run Macro This macro may take a fe
140. ro After installing the capillary secure it into place by pressing a piece of thermal tape over it onto the heat plate just above the electrode Note The capillary should be approximately flush with or less than 1 mm below the end of the electrode Calibrate the autosampler Make sure that it is calibrated in the X Y and Z directions The capillary should almost touch the metal calibration points Refer to Calibrating the Autosampler in the ABI PRISM 310 Genetic Analyzer User s Manual IMPORTANT The sample tray must be removed before calibrating the autosampler If the sample tray is not removed the electrode may bend 3 8 Protocol for 310 Genetic Analyzer with Mac OS Filling the Buffer Reservoirs To flll the buffer reservoirs Step Action 1 Dilute 5 mL of 10X Genetic Analyzer Buffer with EDTA P N 402824 to 1X concentration 50 mL with distilled deionized water Change to fresh buffer every 48 hours or 96 injections whichever comes first a Fill the anode buffer reservoir to the red line with 1X Genetic Analyzer Buffer b Secure the reservoir on the pump block CHEMICAL HAZARD 10X Genetic Analyzer Buffer with EDTA May cause eye skin and respiratory tract irritation Please read the MSDS and follow handling instructions Wear appropriate protective eye wear clothing and gloves a Fill a 4 mL glass buffer vial P N 401955 to the fill line with 1X Geneti
141. rtl D L and Clark A G 1989 Principles of population genetics 2nd edition Sunderland MA Sinauer Associates Inc Hammond H Jin L Zhong Y Caskey C and Chakraborty R 1994 Evaluation of 13 short tandem repeat loci for use in personal identification applications Am J Hum Genet 55 175 189 Holt C Stauffer C Wallin J Lazaruk L Nguyen T Budowle B and Walsh P 2000 Practical applications of genotypic surveys for forensic STR testing Forensic Sci Int 112 2 3 91 109 Holt C et al 2001 TWGDAM validation of AmpF2STR PCR Amplification Kits for Forensic DNA Casework Journal of Forensic Sciences in press Karlin S Cameron E C and Williams P T 1981 Sibling and parent offspring correlation estimation with variable family size Proc Natl Acad Sci USA 78 2664 2668 Kimpton C Walton A and Gill P 1992 A further tetranucleotide repeat polymorphism in the vWF gene Hum Mol Genet 1 287 Kimpton C P Gill P Walton A Urquhart A Millican E S and Adams M 1993 Automated DNA profiling employing multiplex amplification of short tandem repeat loci PCR Methods Appl 3 13 22 Kimpton C P Oldroyd N J Watson S K Frasier R R E Johnson P E Millican E S Urquhart A Sparkes R L and Gill P 1996 Validation of highly discriminating multiplex short tandem repeat amplification systems for human identification Electrophoresis 17 1283 1293 Kinsey
142. running conditions are constant from run to run This is because the spectral overlap between the five dyes is reproducible under constant run conditions However it is recommended that a new matrix be made once a month for use with the AmpFZSTR products or when changing lots of polymer capillaries and buffer Multicomponent analysis is accomplished automatically by the GeneScan Analysis software which applies a mathematical matrix calculation using the values in the matrix file to all sample data Protocol for 310 Genetic Analyzer with Mac OS 3 13 Filter Set G5 Module Files Overview The ABI PRISM9 310 Data Collection Software v2 1 collects light intensities from five specific areas on the CCD camera each area corresponding to the emission wavelength of a particular fluorescent dye Each of these areas on the CCD camera is referred to as a virtual filter since no physical filtering hardware e g band pass glass filter is used The information that specifies the appropriate virtual filter settings for a particular set of fluorescent dyes is contained in each approprate ABI PRISM Data Collection Software module file The module file that must be installed and used for dye set DS 33 6 FAM VIC NED PET LIZ dyes on the ABI PRISM 310 Genetic Analyzer is the following Instrument Configuration Module File ABI PRISM 310 POP 4 polymer GS STR POP4 1 mL G5 with 1 mL syringe IMPORTANT Fi
143. s allele 13 is expected to be found at a size that is 4 bp longer than allele 12 To maximize the ease of peak recognition the size width for most offset categories is 1 bp as compared to the allele categories which have a width of 0 5 bp Once allele 13 is recognized in the D3S1358 allelic ladder the correct offset value is calculated and assigned to the appropriate categories This process of peak recognition offset calculation and offset assignment is carried out for each of the alleles in each of the allelic ladders In the previous example the 12 0s offset value 3 01 is also applied to two other categories in the D3S1358 group OL Allele and allele 11 The OL Allele category is specified to span the range of known D3S1358 alleles and is intended to catch off ladder alleles that do not size within one of the allele categories Allele 11 in this case is a virtual allele category meaning that this allele is not present in the allelic ladder The virtual category exists to assign an allele designation to allele 11 which is a known allele not included in the allelic ladder Because allele 11 is specified to have the same offset value as allele 12 the allele category sizes for these two alleles will differ by exactly 4 bp which is the same as the difference in their reference sizes Specifying a size for allele 11 that is 4 bp shorter than allele 12 is generally expected to be a reasonable estimate since alleles 1
144. samples 3 22 to 3 23 sample electrophoresis 3 24 to 3 25 setting uparun 3 5 to 3 11 setting up the instrument 3 4 to 3 5 using Performance Optimized Polymer 4 for analysis 3 2 to 3 34 agarose gel using to examine DNA 4 35 allele frequencies in the population databases 4 44 to 4 53 AmpF STR Allelic Ladders calculating precision data using the allelic ladders 4 7 to 4 17 using to determine genotypes 5 19 to 5 21 AmpF STR Identifiler loci allele frequencies in the population databases 4 44 to 4 53 Probability of Exclusion Pg from Paternity 4 56 Probability of Identity P 4 55 AmpF STR Identifiler Kit Template 5 3 to 5 16 examining data 5 6 to 5 7 making tables 5 7 to 5 9 troubleshooting genotyping 5 10 understanding the template kit 5 10 to 5 16 using the kit 5 4 to 5 5 amplification differential amplification of loci 4 34 AmpliTaq Gold DNA Polymerase catalyzing the addition of a3 A nucleotide 4 24 automated genotyping about the software 5 2 AmpF STR Identifiler Kit Template examining data 5 6 to 5 7 making tables 5 7 to 5 9 troubleshooting genotyping 5 10 understanding the template kit 5 10 to 5 16 using the kit 5 4 to 5 5 AmpF STR Identifiler Kit Template 5 3 to 5 16 before running Genotyper 5 2 to 5 3 C contamination C 2 customer support See technical support A 1 D degraded DNA 4 34 to 4 35 differential amplification ofloci 4 34 DNA amplification using bloodstained FTA cards 2 9 effect of DNA quantity
145. saster Short tandem repeat mutations observed Progress in Forensic Genetics 8 Eds G F Sensabaugh et al Elsevier Science pp 40 42 Fregeau C J Germain O and Fourney R M 2000 Fingerprint enhancement revisited and the effects of blood enhancement chemicals on subsequent Profiler Plus fluorescent short tandem repeat DNA analysis of fresh and aged bloody fingerprints J Forensic Sci 45 2 354 380 Gill P Urquart A Millican E Oldroyd N Watson S Sparkes R and Kimpton C P 1996 A new method of STR interpretation using inferential logic development of a criminal intelligence database nt J Leg Med 109 14 22 Gill P d Aloja E Andersen J Dupuy B Jangblad M Johnsson V Kloosterman A D Kratzer A Lareu M V Meldegaard M Phillips C Pfitzinger H Rand S Sabatier M Scheithauer R Schmitter H Schneider P and Vide M C 1997 Report of the European DNA Profiling Group EDNAP an investigation of the complex STR loci D21S11 and HUMFIBRA FGA Grossman P D Bloch W Brinson E Chang C C Eggerding F A Fung S lovannisci D M Woo S Win Deen E S 1994 High density miltiplex detection of nucleic acid sequences oligonucleotide ligation assay and sequence coded separation Nucleic Acids Res 22 21 4527 34 Guo S W and Thompson E A 1992 Performing the exact test of Hardy Weinbergproprtion for multiple alleles Biometrics 48 361 372 Ha
146. scale Re amplify the sample if necessary Note DNA samples with off scale data should be diluted and re amplified Protocol for 310 Genetic Analyzer with Mac OS 3 33 Shutting Down the Instrument Ending the Run f the instrument is not going to be in use for 3 or more consecutive days it is recommended that the instrument be cleaned and shut down To shut down the instrument Step Action 1 Remove and clean the syringe and block as previously described 2 Discard unused polymer in the proper waste container Note Do not put unused polymer back into the bottle Polymer in the syringe decomposes over time at room temperature In the Manual Control window select Autosampler Home X Y Axis and click Execute Select Autosampler Home Z Axis and click Execute Turn off the instrument 3 34 Protocol for 310 Genetic Analyzer with Mac OS Dedicated Equipment and Supplies Equipment The following are equipment and supplies necessary or recommended Required for running AmpF STR Identifiler kit data on the ABI PRISM 310 Genetic Analyzer Note Amplified DNA equipment and supplies used to handle amplified DNA should not be taken out of the amplified DNA work area Samples that have not yet been amplified should never come into contact with these supplies and equipment Equipment Required Equipment ABI PRISM 310 Genetic Analyzer mL P N 401955 ABI
147. sign genotypes to those sample allele peaks falling within 0 5 bp of the corresponding allelic ladder peak The allele designation for each allelic ladder peak is given in Figure 5 1 on page 5 18 The AmpF STR Identifiler Allelic Ladder contains the majority of alleles for the Amelogenin CSF1PO D281338 D381358 D5S818 D7S820 D881179 D13S317 D16S539 D18S51 D19S433 D21S11 FGA TH01 TPOX and vWA loci However alleles not found in the 5 20 Genotyping for the Macintosh OS AmpFZSTR Identifiler Allelic Ladder do exist These off ladder alleles may contain full and or partial repeat units An off ladder allele should flag itself by not falling inside the 0 5 bp window of any known allelic ladder allele Note Ifa sample allele peak is found to be 20 5 bp from the corresponding allelic ladder peak then the sample must be rerun to verify the result Genotyping for the Macintosh OS 5 21 Technical Support Contacting Technical Support To Contact Technical Support by E Mail You can contact Applied Biosystems for technical support by telephone or fax by e mail or through the Internet You can order Applied Biosystems user documents MSDSs certificates of analysis and other related documents 24 hours a day In addition you can download documents in PDF format from the Applied Biosystems Web site please see the section To Obtain Documents on Demand following the telephone information below Conta
148. ssignments are incorrect for one injection define a new size standard for that sample using the peaks in that injection To do so select the Define New option in the Size Standard pop up menu for that sample Re analyze any incorrectly sized samples select the blue green yellow red and orange boxes using the newly defined GeneScan 500 LIZ Size Standard file This creates a new standard file for each of these samples replacing the previous analysis results for those samples only 3 28 Protocol for 310 Genetic Analyzer with Mac OS To set the analysis parameters continued Step Action 10 Confirm that the GeneScan 500 LIZ Size Standard peaks are now correctly assigned in the re analyzed samples 11 View AmpFZSTR Identifiler kit results using the Results Control window Refer to the GeneScan Analysis 3 1 User s Manual for printing options Protocol for 310 Genetic Analyzer with Mac OS 3 29 GeneScan Software Results GeneScan Analysis Software Information Provided in the Electropherogram and Table After the sample files have been analyzed the Results Control window is used to display the results from each lane of a gel or each injection into a capillary The Results Control window displays the newly analyzed sample files and allows the user to specify the format of the results Selecting both the Electropherogram and Tabular Data icons is recommended for reviewing the resul
149. sterolemia Nat Biotechnol 14 10 1279 82 References F 1 F 2 References Begovich A B McClure G R Suraj V C Helmuth R C Fildes N Bugawan T L Erlich H A and Klitz W 1992 Polymorphism recombination and linkage disequilibrium within the HLA Class II region J Immunol 148 249 258 Brinkmann B Moller A and Wiegand P 1995 Structure of new mutations in 2 STR systems Int J Legal Med 107 201 203 Brinkmann B Junge A Meyer E and Wiegand P 1998 Population genetic diversity in relation to microsatellite heterogeneity Hum Mutat 11 135 144 Brinkmann B Klintschar M Neuhuber F Huhne J and Rolf B 1998 Mutation rate in human microsatellites Influence of the structure and length of the tandem repeat Am J Hum Genet 62 1408 1415 Brown A H D Feldman M W and Nevo E 1980 Multilocus structure of natural populations of Hordeum spontaneum Genetics 96 523 536 Budowle B et al 1995 D1S80 population data in African Americans Caucasians Southeastern Hispanics Southwestern Hispanics and Orientals J Forensic Sci 40 38 44 Budowle B et al 1998a CODIS and PCR Based Short Tandem Repeat Loci Law Enforcement Tools Second European Symposium on Human Identification 73 88 Budowle B Baechkel F Fejeren R 1998b Polymarker HLA DQAQ and D1S80 allele frequency data in Chamorro and Filipino populations from Guam Journal Forensic Science 43 6 1195 1198
150. t its maximum peak height the data point correlates with the number of laser scans or data points collected from the beginning of the run until the time that the peak maximum is detected 3 30 Protocol for 310 Genetic Analyzer with Mac OS i HD Dye Sample Minutes Size Peak Height Peak Area Data Point Figure 3 6 GeneScan electropherogram of AmpFZSTR Identifiler alleles in AmpFZSTR Control DNA 9947A analyzed on the ABI PRISM 310 Genetic Analyzer Results Display Options The GeneScan Software v3 1 or higher offers two main options in the Results Control window for electropherogram viewing formats Quick Tile Off and Quick Tile On The Quick Tile Off format provides the option of displaying results either for multiple colors within a single lane or injection or from multiple lanes or injections in the same panel i e the results are overlaid This is demonstrated in panel 1 of Figure 3 7 The Quick Tile On format displays each color of each lane or injection separately as shown in panels 2 5 of Figure 3 7 The Quick Tile Off and On feature offers the user versatility in customizing the display of results Up to eight panels can be tiled at a single time and up to 16 electropherogram may be overlaid in one panel at the same time Protocol for 310 Genetic Analyzer with Mac OS 3 31 180 300 320 340 11 illu li i
151. t listed 44 0 1925 282481 44 0 1925 282509 Warrington UK Japan Japan Hacchobori 81 20 477392 Toll 81 20 477120 Toll Chuo Ku Tokyo free or free or 81 3 5566 6230 81 3 5566 6507 Latin America Caribbean countries 52 55 35 3610 52 55 66 2308 Mexico and Central America Brazil 0 800 704 9004 or 55 11 5070 9694 95 55 11 5070 9654 Argentina 800 666 0096 55 11 5070 9694 95 Chile 1230 020 9102 55 11 5070 9694 95 Uruguay 0004 055 654 55 11 5070 9694 95 We strongly encourage you to visit our Web site for answers to frequently asked questions and for more information about our products You can also order technical documents or an index of available documents and have them faxed or e mailed to you through our site The Applied Biosystems Web site address is http www appliedbiosystems com techsupp To submit technical questions from North America or Europe Step Action 1 Access the Applied Biosystems Technical Support Web site 2 Under the Troubleshooting heading click Support Request Forms then select the relevant support region for the product area of interest 3 In the Personal Assistance form enter the requested information and your question then click Ask Us RIGHT NOW 4 In the Customer Information form enter the requested information and your question then click Ask Us RIGHT NOW Within 24 to 48 hours you will receive an e mail reply to your q
152. tandards for Parentage Testing Laboratories Amorin A Alves C Gusmao L 2000 Somatic and Germinal Mutations in STRs Progress in Forensic Genetics 8 Eds G F Sensabaugh et al Elsevier Science pp 37 39 Andersen J F Greenhalgh M J Butler H R Kilpatrick S R Piercy R C Way K A Myhill H S Wright J C Hallett R and Parkin B H 1996 Further validation of a multiplex STR system for use in routine forensic identity testing Forensic Sci Int 78 47 64 B r W Brinkmann B Budowle B Carracedo A Gill P Lincoln P Mayr W and Olaisen B 1997 DNA recommendations Further report of the DNA Commission of the ISFH regarding the use of short tandem repeat systems Intl J Legal Med 110 175 176 Barber M D Piercy R C Andersen J F and Parkin B H 1995 Structural variation of novel alleles at the Hum vWA and Hum FES FPS short tandem repeat loci Intl J Legal Med 108 31 35 Barber M D McKeown B J and Parkin B H 1996 Structural variation in the alleles of a short tandem repeat system at the human alpha fibrinogen locus Intl J Legal Med 108 180 185 Barber M D and Parkin B H 1996 Sequence analysis and allelic designation of the two short tandem repeat loci D18S51 and D8S1179 Intl J Legal Med 109 62 65 Baron H Fung S Aydin A Bahring S Luft F C Schuster H 1996 Oligonucleotide ligation assay OLA for the diagnosis of familial hyperchole
153. th the AmpF STR Identifiler PCR Amplification Kit is provided with this manual and should be used with AmpFZSTR Identifiler kit data Install the template onto your computer following the instructions in the READ ME file Note You must have Genotyper Software v2 5 2 or higher to run the AmpF4STR Identifiler Kit Template It is recommended that this version of Genotyper software be run on a Power Macintosh computer with Macintosh OS 8 x or 9 1 Refer to the Genotyper Software User s Manual Electronic Document 904648 and Genotyper Applications Tutorials Electronic Document 904649 for more detailed information about the Genotyper software The Human Identification Tutorial and HID template file included with the Genotyper software v2 5 2 software package are for tutorial purposes only GeneScan Analysis Software sample data particularly the allelic ladder must meet a few specific requirements before the macros in the AmpFZSTR Identifiler Kit Template can be used These requirements are described in this section Sample Info All samples must have a unique sample description in the Sample Info column of the GeneScan software sample sheet so that the macros in the AmpFZSTR Identifiler Kit Template can build a table Samples with an empty Sample Info field will not be incorporated into the table of genotypes Also lanes or injections that contain the AmpFZSTR Identifiler Allelic Ladder must have the word ladder in the Sample Info The
154. the GeneScan software table Sample allele peak heights must also be greater than the GeneScan Software PAT in order to be recognized labeled by Genotyper software Note that the PAT value specified in the GeneScan software Analysis Parameters is not necessarily the same as the RFU value that may be used by the forensic analyst as the interpretational threshold The Low Signal column of the appropriate Genotyper software table see page 5 8 can be used to identify peaks that are greater than the GeneScan software PAT but less than a specified minimum threshold default 150 RFU in the table macro AmpF STR The AmpFZSTR Identifiler Kit Template contains macros that perform Identifiler Kit the following steps automatically Template 4 Finds the lane or injection containing the allelic ladder Creates allele size categories that are centered on the sizes obtained for the allelic ladder alleles Assigns the appropriate allele label to sample alleles that size within the allele size categories Removes labels from stutter peaks by applying a filter Builds a table containing genotypes for all samples Genotyping for the Macintosh OS 5 3 Using the Use the following procedure to assign genotypes to AmpFZSTR AmpFE STR dentifiler kit alleles automatically Identifiler Kit Template File To use the AmpF STR Identifiler Kit Template Step Action 1 Double click the IDENTIFILER icon to launch the Genotyper softwa
155. the loci amplified and the corresponding dyes the Kit used Amplified Loci Alleles Included in Chromosome Identifiler Allelic Control Locus Designation Location Ladder Dye Label DNA 9947A D881179 8 8 9 10 11 12 13 6 FAM 13a 14 15 16 17 18 19 D21S11 21q11 2 q21 24 24 2 25 26 27 30b 28 28 2 29 29 2 30 30 2 31 31 2 32 32 2 33 33 2 34 34 2 35 35 2 36 37 38 D7S820 7q11 21 22 6 7 8 9 10 11 12 10 11 13 14 15 CSF1PO 5q33 3 34 6 7 8 9 10 11 12 10 12 13 14 15 D381358 3p 12 18 14 15 16 VIC 14 15 17 18 19 THO1 11p15 5 4 5 6 7 8 9 9 3 8 9 3 10 11 13 3 D13S317 13q22 31 8 9 10 11 12 13 11 14 15 D16S539 16q24 qter 5 8 9 10 11 12 13 11 12 14 15 D281338 2q35 37 1 15 16 17 18 19 19 23 20 21 22 23 24 25 26 27 28 1 4 Introduction Amplified Loci continued Chromosome Locus Designation Location Alleles Included in Identifiler Allelic Ladder Dye Label Control DNA 9947A D19S433 19q12 13 1 9 10 11 12 12 2 13 13 2 14 14 2 15 15 2 16 16 2 17 17 2 vWA 12p12 pter 11 12 13 14 15 16 17 18 19 20 21 22 23 24 TPOX 2p23 2per 6 7 8 9 10 11 12 13 D18S51 18q21 3 7 9 10 10 2 11 12 13 13 2 14 14 2 15 16 17 18 19 20 21 22 23 24 25 26 27 NED 14 15 17 18
156. ting step occurs automatically at the beginning of the GS STR POP4 1 mL G5 run module To set the run temperature Step Action 1 Close the instrument doors 2 Return to the ABI PRISM 310 Collection Software 3 a From the Window menu select Manual Control b Select Temperature Set from the pop up menu 4 a Set the temperature to 60 C b Click Execute Note It takes up to 30 min for the instrument to reach the 60 C run temperature Samples can be prepared while the instrument is heating Setting the To choose a five dye sample sheet Parameters Step Action 1 This is an optional step Launch the 310 Genetic Analyzer Data Collection software v2 1 From the Window menu under Preferences choose the GeneScan Sample Sheet Defaults Set the size standard color to orange O as shown in the figure Preferences Page GeneScan Sample Sheet Defaults t B G Y R 0 Size Standard Dye Color L Cx Protocol for 310 Genetic Analyzer with Mac OS 3 11 To choose a five dye sample sheet continued 2 Select GeneScan Injection List Defaults from the Page drop down menu The following window appears Preferences Page GeneScan Injection List Defaults t Length to Detector cm rg 7 4Dye Module none Z 5 Dye Module none Z M
157. tion of these artifacts as off ladder alleles or OL Alleles Note the degree of magnification y axis used in this figure to illustrate these artifacts data produced on the ABI PRISM 310 Genetic Analyzer Artifacts can be intermittent and are not always reproducible In our experience non reproducible artifacts can be correlated to sources other than the kit e g spikes An intermittent artifact is not observed in the same position upon re injection The Identifiler kit user should consider these artifacts when interpreting data Experiments and Results 4 27 Characterization of Loci 8 1 2 1 Documentation exists and is available which defines and characterizes the Documentation ocus DAB 1998 Overview This section describes basic characteristics of the 16 loci that are amplified with the AmpF STR Identifiler kit These loci have been previously characterized Nature of the The primers for the Amelogenin locus flank a six base pair deletion Polymorphisms within intron 1 of the X homologue Amplification results in 107 bp and 113 bp products from the X and Y chromosomes respectively Sizes are the actual base pair size according to sequencing results including 3 A nucleotide addition The remaining AmpFZSTR Identifiler kit loci are all tetranucleotide short tandem repeat STR loci The length differences among alleles of a particular locus result from differences in the number of 4 bp repeat units Some alleles in
158. to designated areas In This Appendix Appendix C contains the following topics Topic See Page Overview C 1 Lab Design C 2 Laboratory Setup C 1 Lab Design Sensitivity of PCR Many resources are available for the appropriate design of a PCR C 2 Laboratory Setup laboratory If you are using the Identifiler kit for forensic DNA testing you may want to refer to http www ojp usdoj gov nij scidocs htm Forensic Laboratories Handbook for Facility Planning Design Construction and Moving If you are using the Identifiler kit for parentage DNA testing you may want to refer to the Standards for Parentage Testing Laboratories The sensitivity of the AmpF STR Identifiler PCR Amplification Kit and other PCR based tests permits amplification of minute quantities of DNA This necessitates precautions to avoid contamination of samples yet to be amplified Kwok and Higuchi 1989 While contamination of amplified DNA with unamplified DNA genomic DNA does not pose a problem ordinary precautions such as changing pipet tips between samples should be taken when handling and analyzing PCR product These precautions should prevent cross contamination between samples of amplified DNA Care should be taken while handling and processing samples to prevent chance contamination by human DNA Gloves should be worn at all times and changed frequently Sample tubes should be closed when not in use Dispersal of aeroso
159. to non nucleotide linkers Grossman et al 1994 and Baron et al 1996 The non nucleotide linkers enable reproducible positioning of the alleles to facilitate inter locus spacing The loci which incorporate these non nucleotide linkers are as follows CSF1PO D281338 D13S317 D16S539 and TPOX The AmpFZSTR Identifiler PCR Amplification Kit is designed so that a majority of the PCR products contain the non templated 3 A nucleotide The alleles have been named in accordance with the recommendations of the DNA Commission of the International Society for Forensic Haemogenetics ISFH DNA Recommendations 1994 Bar et al 1997 The number of complete four base pair repeat units observed is designated by an integer Variant alleles that contain a partial repeat are designated by a decimal followed by the number of bases in the partial repeat For example an FGA 26 2 allele contains 26 complete repeat units and a partial repeat unit of two base pairs Genotyping for the Macintosh OS 5 17 Additional variation has been seen at some loci where alleles exist that differ from integer allele lengths by one or three base pairs For example D21S11 allele 33 1 contains 33 complete repeat units and one nonconsensus base pair Likewise a D21811 29 3 allele contains 29 complete repeat units and a partial 3 bp unit Moller et a 1994 Gill et al 1997 A Genotyper software electropherogram of the AmpFZSTR Identifiler Allelic Ladder listing the desi
160. trol DNA 9947A AmpF4STR Identifiler Allelic Ladder AmpliTaq Gold DNA Polymerase 15 to 25 C Introduction 1 7 Safety Documentation User Attention Words Chemical Hazard Warning 1 8 Introduction Five user attention words appear in the text of all Applied Biosystems user documentation Each word implies a particular level of observation or action as described below Note Calls attention to useful information IMPORTANT Indicates information that is necessary for proper instrument operation yx e Indicates a potentially hazardous situation which if not avoided may result in minor or moderate injury It may also be used to alert against unsafe practices AGANE Indicates a potentially hazardous situation which if not avoided could result in death or serious injury PN eldi Indicates an imminently hazardous situation which if not avoided will result in death or serious injury This signal word is to be limited to the most extreme situations Ete CHEMICAL HAZARD Some of the chemicals used with Applied Biosystems instruments and protocols are potentially hazardous and could cause injury illness or death Readand understand the material safety data sheets MSDSs provided by the chemical manufacturer before you store handle or work with any chemicals or hazardous materials Minimize contact with chemicals Wear appropriate personal protective equipment when handling chemicals e g safety gl
161. ts For more information on displaying the results refer to the GeneScan Analysis Software v3 1 User s Manual P N 903565 Both the electropherogram and the tabular data can be displayed see Figure 3 6 on page 3 31 The electropherogram is a chromatographic display with fluorescence intensity indicated as relative fluorescence units RFU on the y axis After the internal lane size standard has been defined and applied the electropherogram can be displayed with the base pair size on the x axis Peaks of all heights within the Analysis Range specified in the Analysis Parameters are displayed on the electropherogram but those peaks below the Peak Amplitude Threshold minimum peak height that are defined in the Analysis Parameters will not be listed in the tabular data The columns in the table list the following Column 1 lists the Dye Sample and Peak e g 4B 1 indicates the first blue peak in project sample 4 Column 2 lists the time it took the dye labeled fragment to reach the detector Column 3 lists the base pair size of the peak as calculated using the GeneScan 500 LIZ Size Standard curve Column 4 lists the height in RFU of the peak Column 5 lists the relative peak area which is the integral of the RFU times the data point scan number This value depends on the velocity of the dye labeled fragment as it passes the detector Column 6 lists the data point scan number of the dye labeled fragment a
162. turation temperature experiments Of the tested annealing temperatures 55 57 59 and 61 C produced robust profiles At 63 C the yield of the majority of loci was significantly reduced This should pose no problem with routine thermal cycler calibration and when following the recommended amplification protocol Preferential amplification was not observed at any of the tested annealing temperatures 4 4 Experiments and Results 55 C 2400 i ll dia abad La m nada 2 2400 59 C e usd T da usun aem 2400 EE E ti 0 la adh asandi A 61 C 2400 1200 63 C Figure 4 2 An amplification of 1 ng of genomic DNA amplified while varying the annealing temperature analyzed on the ABI PRISM 310 Genetic Analyzer AmpliTaq Gold DNA Polymerase Activation PCR Cycle Number The thermal cycler program for the AmpF STR Identifiler kit includes an initial incubation to allow for AmpliTaq Gold DNA polymerase activation Polymerase activation times and temperatures were tested in the GeneAmp PCR Systems 9600 and 9700 Five activation times 5 8 11 14 and 17 minutes were tested previously for the AmpF STR kits Wallin et al 1998 A plateau in relative fluorescent signal RFU of the AmpFZSTR Identifiler kit loci was reached at approximately 8 minutes and was maintained through the 17 minute time point The 11 minute activation time was determined to be optimal based on this plateau
163. uestion from an Applied Biosystems technical expert Technical Support A 5 To Obtain Free 24 hour access to Applied Biosystems technical documents Documents on including MSDSs is available by fax or e mail or by download from our Demand Web site To order documents Then by index a Access the Applied Biosystems Technical Support Web number site at http www appliedbiosystems com techsupp b Click the Index link for the document type you want then find the document you want and record the index number c Use the index number when requesting documents following the procedures below by phone for a From the U S or Canada call 1 800 487 6809 or fax delivery from outside the U S and Canada call 1 858 712 0317 b Follow the voice instructions to order the documents you want Note There is a limit of five documents per request through the a Access the Applied Biosystems Technical Support Web Internet for fax site at or e mail http www appliedbiosystems com techsupp delivery b Under Resource Libraries click the type of document you want c Enter or select the requested information in the displayed form then click Search d In the displayed search results select a check box for the method of delivery for each document that matches your criteria then click Deliver Selected Documents Now or click the PDF icon for the document to download it immediately e Fill in th
164. unused polymer to the bottle Note Before use the POP 4 polymer should be allowed to equilibrate to room temperature If precipitate is present in the bottle when removed from cold storage it should go back into solution at room temperature Gently mix the polymer thoroughly by inversion before using 3 Wipe the outside of syringe with a Kim wipe tissue to dry 4 Remove any air bubbles by inverting the syringe and pushing a small amount of polymer out of the tip Removing and Cleaning the Pump Block Before setting up the instrument for a run make sure that the pump block is clean of all polymer especially if the polymer in the syringe has been sitting at room temperature for more than three days Urea decomposition during this interval causes transient current increases spikes during electrophoresis To remove and clean the pump block see Cleaning and Maintaining the Instrument in the AB PRISM 310 Genetic Analyzer User s Manual Follow the instructions in the sections titled Removing the Pump Block Rinsing the Pump Block and Replacing the Pump Block We do not recommend following the section titled Rinsing the Pump Block on the Instrument for this application IMPORTANT Remove all residual water from the pump block and fittings by blowing canned compressed air through the channels Make sure the can is held upright or the propellant in the can may be shot into the gel block This can result in poo
165. utation was represented by an increase of one 4 bp repeat unit a 17 allele was inherited as an 18 single step mutation The maternal paternal source of this mutation could not be distinguished Additional studies Edwards et a 1991 Edwards et al 1992 Weber and Wong 1993 Hammond et a 1994 Brinkmann et al 1995 Chakraborty et al 1996 Chakraborty et a 1997 Brinkmann et al 1998 Momhinweg et a 1998 Szibor et a 1998 of direct mutation rate counts produced Larger sample sizes for some of the AmpF STR Identifiler kit loci Methods for modifications of these mutation rates to infer mutation rates indirectly for those loci where these rates are not large enough to be measured directly and or to account for those events undetectable as Mendelian errors Experiments and Results 4 55 Probability of Identity Table of Table 4 4 shows the Probability of Identity Pj values of the AmpFZSTR Probability of dentifiler kit loci individually and combined Identity Table 4 4 Probability of Identity values for the AmpFZSTR Identifiler kit STR loci Locus African American U S Caucasian U S Hispanic Native American CSF1PO 0 079 0 132 0 141 0 123 D2S1338 0 023 0 027 0 038 0 043 D3S1358 0 097 0 076 0 112 0 158 D5S818 0 104 0 147 0 115 0 110 D7S820 0 085 0 063 0 083 0 081 D8S1179 0 074 0 064 0 089 0 104 D13S317 0 132 0 079 0 056 0 056 D16S539 0 077 0 097 0 090 0 082 D18S51 0 033 0 031 0 031 0 046 D19S433 0 042 0 087
166. veal phylogenetic relationships in primates J Mol Evol 41 1 10 4 Mills K A Even D and Murray J C 1992 Tetranucelotide repeat polymorphism at the human alpha fibrinogen locus FGA Hum Mol Genet 1 779 Moller A Meyer E and Brinkmann B 1994 Different types of structural variation in STRs HumFES FPS HumVWA and HumD21811 Intl J Legal Med 106 319 323 Moller A and Brinkmann B 1995 PCR VNTRs PCR Variable Number of Tandem Repeats in forensic science Cell Molec Biol 41 715 724 Momhinweg E Luckenbach C Fimmers R and Ritter H 1998 D3S1358 sequence analysis and gene frequency in a German population Forensic Sci Int 95 173 178 Nakahori Y Takenaka O and Nakagome Y 1991 A human X Y homologous region encodes amelogenin Genomics 9 264 269 National Research Council 1996 The evaluation of forensic DNA evidence National Academy Press Washington D C Nei M and Roychoudhury A K 1974 Sampling variances of heterozygosity and genetic distance Genetics 76 379 390 Nei M 1978 Estimation of average heterozygosity and genetic distance from a small number of individuals Genetics 89 583 590 Oldroyd N J Urquhart A J Kimpton C P Millican E S Watson S K Downes T and Gill PD 1995 A highly discriminating octoplex short tandem repeat polymerase chain reaction system suitable for human individual identification Electrophoresis 16 334 337 Opp
167. volume of 10 uL final sample concentration is 0 05 0 125 ng uL Positive Control Tube a Vortex the AmpFL STR Control DNA 9947A tube 0 10 ng uL b Spin the tube briefly in a microcentrifuge to remove any liquid from the cap c Add 10 uL 1 ng of AmpFZSTR Control DNA 9947A to the Positive Control Tube Negative Control Tube Add 10 uL of TE buffer see page 2 4 for preparation to the labeled Negative Control Tube Performing PCR Thermal Cyclers Use either of the following thermal cyclers to amplify loci using the AmpF4ZSTR Identifiler kit GeneAmp PCR System 9700 GeneAmp PCR System 9600 Amplifying the To amplify the DNA DNA Step Action 1 Program the thermal cycling conditions IMPORTANT If using the GeneAmp PCR System 9700 select the 9600 Emulation Mode Initial Incubation Final Final Step Denature Anneal Extend Extension Step HOLD CYCLE 28 cycles HOLD HOLD 95 C 94 C 59 C 72 C 60 C 4 25 C 11 min 1 min 1 min 1 min 60 min forever Note If leaving the amplified products in the thermal cycler for more than 18 hr set the final step to HOLD at 4 25 C forever The final step can be held anywhere in this range Each laboratory should determine the final time and temperature to store PCR products in the thermal cycler Place the tray in the thermal cycler Close the heated cover Start the thermal cycle
168. w minutes to run When it is finished a plot window opens with the blue allelic ladder D8S1179 D21S11 D7S820 and CSF1PO and sample allele peaks labeled Examine data and edit peaks Print the electropherograms in the plot window by choosing Print from the File menu 5 4 Genotyping for the Macintosh OS To use the AmpFZSTR Identifiler Kit Template continued Step Action 11 In the Main Window click the green G button at the top left From the Views menu select Show Plot Window Repeat steps 8 and 9 12 In the Main Window click the yellow Y button at the top left From the Views menu select Show Plot Window Repeat steps 8 and 9 13 In the Main Window click the red R button at the top left From the Vlews menu select Show Plot Window Repeat steps 8 and 9 9 FT M O FT M O D W Genotyping for the Macintosh OS 5 5 Examining Data Check that the peaks in the allelic ladder are labeled correctly Scroll through the samples below the allelic ladder to examine the peak labels in each electropherogram Peak Labeling Allele categories which appear as dark gray bars in the Plot window are defined to be 0 5 bp wide Peaks that size within x 0 5 bp of an allele category will have a label indicating the allele designation Note The categories for THO1 alleles 9 3 and 10 are 0 4 bp wide Peaks that do not size within an allele category will have a label indic
169. within a gt 25 window AmpFZ4STR ldentifiler kit reactions were amplified for 27 28 29 30 and 31 cycles on the GeneAmp PCR System 9700 using 1 0 ng of three DNA samples As expected PCR product increased with the number of cycles A full profile was generated at 27 cycles off scale data were collected for several allele peaks at 31 cycles While none of the cycle numbers tested produced nonspecific peaks 28 cycles was found to give optimal sensitivity when the amplified products were examined on ABI PRISM 310 Genetic Analyzers Additionally the cycle number was set to avoid detection of low quantities of DNA 20 pg or less At 28 cycles 1 0 ng of AmpFZSTR Control DNA 9947A amplifies reliably and specifically following the conditions outlined in this user manual Experiments and Results 4 5 Accuracy Precision and Reproducibility 8 1 2 Accuracy Size Deviation bp Novel forensic DNA methodologies shall undergo developmental validation to ensure the accuracy precision and reproducibility of the procedure DAB 1998 Laser induced fluorescence detection systems of length polymorphism at short tandem repeat loci is not a novel methodology Holt et a 2001 and Wallin et al 2001 However accuracy and reproducibility of AmpFZSTR Identifiler kit profiles have been determined from various sample types Figure 4 3 illustrates the size differences that are typically observed between sample alleles and allelic ladd
170. x not vortexed thoroughly before aliquoting Vortex PCR Master Mix thoroughly AmpFZSTR Identifiler Primer Set exposed to too much light Store Primer Set protected from light GeneAmp PCR System malfunction Refer to the thermal cycler user s manual and check instrument calibration Incorrect thermal cycler parameters Check the protocol for correct thermal cycler parameters Tubes not seated tightly in the thermal cycler during amplification Push reaction tubes firmly into contact with block after first cycle Repeat test GeneAmp PCR System 9600 heated cover misaligned Align GeneAmp 9600 heated cover properly so that white stripes align after twisting the top portion clockwise Wrong PCR reaction tube MicroAmp Base used with tray retainer set and tubes in GeneAmp 9600 and 9700 Use Applied Biosystems MicroAmp Reaction Tubes with Caps for the GeneAmp 9600 and 9700 Remove MicroAmp Base from tray retainer set and repeat test Insufficient PCR product electrokinetically injected For ABI Prism 310 runs Mix 1 5 uL of PCR product and 24 5 uL of Hi Di Formamide GeneScan 500 LIZTM solution Degraded formamide Check the storage of formamide do not thaw and re freeze multiple times Try Hi Di Formamide B 2 Troubleshooting Observation Positive signal from AmpF4STR Control DNA 9947A but no signal from DNA test samples More t
171. y exists that not every locus will amplify This is most often observed when the DNA sample contains PCR inhibitors or when the DNA sample has been severely degraded Since each locus is an independent marker whose results are not based upon information provided by the other markers results generally can still be obtained from the loci that do amplify Differential amplification can be defined as the difference in the degree of amplification of each locus within a co amplified system such that one or more loci may amplify to a greater extent compared to the other loci Preferential amplification is used in this manual to describe differences in the amplification efficiency of two alleles at a single locus Preferential amplification of alleles in systems that distinguish alleles based on length polymorphisms is most likely to be observed when the alleles differ significantly in base pair size Since most AmpF STR Identifiler kit loci have small size ranges the potential for preferential amplification of alleles is low Heme compounds have been identified as PCR inhibitors in DNA samples extracted from bloodstains DeFranchis et a 1988 Akane et al 1994 It is believed that the inhibitor is co extracted and co purified with the DNA and subsequently interferes with PCR by inhibiting polymerase activity Bovine serum albumin BSA can prevent or minimize the inhibition of PCR most likely by binding to the inhibitor Comey et al 1994 S
172. y only when the instrument is first set up or if the electrode was or has been bent severely IMPORTANT Anew electrode must be trimmed to the correct length Refer to Figure 3 2 for trimming the electrode Note Not all electrodes need to be trimmed Trim only as needed To install a new electrode Step Action 1 Install the new electrode on the instrument as described in the ABI PRISM 310 Genetic Analyzer User s Manual Under the Manual Control menu select Home Z Axis a Use the flush cutting wire cutter P N T 6157 provided in the instrument packing kit b Hold the cutters with the flat cutting face toward the top of the instrument 4 Cut a small amount off the end of the electrode until it is flush with the lower surface of the stripper plate Be careful not to flex the stripper plate upwards while cutting Do not cut off more than 1mm beyond the lower surface of the stripper plate Figure 3 2 MAXIMUM TRIM LENGTH MINIMUM TRIM LENGTH FLUSH WITH BOTTOM imm UP FROM LOWER OF STRIPPER PLATE SURFACE OF STRIPPER TE ios IH m CAPILLARY ELECTRODE x STRIPPER PLATE 1 2 3 4 5 6 Figure 3 2 Trimming the electrode 3 4 Protocol for 310 Genetic Analyzer with Mac OS Cleaning the Electrode To clean the electrode Step Action 1 Wipe the electrode with a Kim wipe tissue that has been d

AmpFlSTR® Identifiler™ PCR Amplification Kit

Contents

Download Pdf Manuals

Related Search

Related Contents

AmpFlSTR&reg; Identifiler&trade; PCR Amplification Kit

Contents

Download Pdf Manuals

Related Search

Related Contents

AmpFlSTR® Identifiler™ PCR Amplification Kit