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PowerPlex® 1.2 System

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1. ramp 100 to 90 C for 30 seconds ramp 100 to 60 C for 30 seconds ramp 23 to 70 C for 45 seconds for 20 cycles then 60 C for 30 minutes 4 C soak Protocol for the Perkin Elmer Thermal Cycler Model 480 95 C for 11 minutes then 96 C for 2 minutes then 94 C for 1 minute 60 C for 1 minute 70 C for 1 5 minutes for 10 cycles then 90 C for 1 minute 60 C for 1 minute 70 C for 1 5 minutes for 20 cycles then 60 C for 30 minutes 4 C soak Q When using Taq DNA polymerase instead of AmpliTaq Gold DNA polymerase do not include the incubation at 95 C for 11 minutes prior to initiating the thermal cycling program Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Telephone 608 274 4330 Fax 608 277 2516 www promega com Part TMD009 Page 10 Printed in USA Revised 6 07 V C Agarose Gel Electrophoresis of Amplification Products Optional Agarose gel electrophoresis can be used to rapidly confirm the success of the amplification reaction prior to performing polyacrylamide gel or capillary electrophoresis Materials to Be Supplied by the User Solution compositions are provided in Section XII A TAE 1X buffer agarose 5X loading solution ethidium bromide solution 0 5ug ml 1 Prepare a 2 agarose gel approximately 150cm by adding 2 0g of agarose to 100ml of TAE 1X buffer Mark the liquid level on the container then boil or
2. analysis parameters Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Telephone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD009 Revised 6 07 Page 29 xI References 1 Edwards A et al 1991 DNA typing with trimeric and tetrameric tandem repeats Polymorphic loci detection systems and population genetics In The Second International Symposium on Human Identification 1991 Promega Corporation 31 52 Edwards A et al 1991 DNA typing and genetic mapping with trimeric and tetrameric tandem repeats Am J Hum Genet 49 746 56 Edwards A et al 1992 Genetic variation at five trimeric and tetrameric tandem repeat loci in four human population groups Genomics 12 241 53 Warne D et al 1991 Tetranucleotide repeat polymorphism at the human D actin related pseudogene 2 actbp2 detected using the polymerase chain reaction Nucl Acids Hes 19 6980 Ausubel F M et al 1993 Unit 15 The polymerase chain reaction In Current Protocols in Molecular Biology Vol 2 Greene Publishing Associates Inc and John Wiley and Sons NY Sambrook J Fritsch E F and Maniatis T 1989 Chapter 14 In vitro amplification of DNA by the polymerase chain reaction In Molecular Cloning A Laboratory Manual Second Edition Cold Spring Harbor Laboratory Press Cold opring Harbor New York PCR Technology Principles and Applicati
3. Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Telephone 608 274 4330 Fax 608 277 2516 www promega com Part TMD009 Page 16 Printed in USA Revised 6 07 4 The analysis parameters can be saved in the Params folder Apply the stored analysis parameters file to the samples Assign a new size standard Select a sample file highlight the arrow next to the size standard then select define new Assign the size standard peaks as shown in Figure 1 Store the size standard in the SizeStandards folder Note If pull up or bleedthrough from TMR yellow is seen in the CXR red channel the peak amplitude threshold can be increased i e 100 200RHFU for the red channel in the analysis parameters This does not interfere with interpretation as the CXR ILS fragments should be greater than 300RFU 7 Apply the size standard file to the samples then analyze the sample files 8 See Section IX for further data analysis For additional information regarding the GeneScan analysis software refer to the GeneScan Analysis Software User s Manual Notes e Amplified sample peak heights 2 000HFU are ideal e Peak heights 2 000HFU might generate artifact peaks due to instrument saturation i e overloading the sample Bleedthrough pull ups or oversub traction pull downs from one color to another might be observed Saturated signal might also appear as two small peaks e If the sample peak heights
4. terization of stutter products at the tetranucleotide repeat locus vWA Nucl Acids Res 24 2807 12 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Telephone 608 274 4330 Fax 608 277 2516 www promega com Part TMD009 Page 30 Printed in USA Revised 6 07 18 Moller A Meyer E and Brinkmann B 1994 Different types of structural varia tion in STRs HumFES FPS HumVWA and HumD21 11 nt J Leg Med 106 319 23 19 Brinkmann B Moller A and Wiegand P 1995 Structure of new mutations in 2 STR systems Int J Leg Med 107 201 3 20 Lins A M et al 1998 Development and population study of an eight locus short tandem repeat STR multiplex system J Forensic Sci 43 1168 80 21 Puers C et al 1993 Identification of repeat sequence heterogeneity at the poly morphic STR locus HUMTHO1 AATG n and reassignment of alleles in population analysis using a locus specific allelic ladder Am J Hum Genet 53 953 8 22 Hammond H et al 1994 Evaluation of 13 short tandem repeat loci for use in personal identification applications Am J Hum Genet 55 175 89 23 Bever R A and Creacy S 1995 Validation and utilization of commercially avail able STR multiplexes for parentage analysis In Proceedings from the Fifth Inter national Symposium on Human Identification 1994 Promega Corporation 61 8 24 Sprecher C J et al 1996 A genera
5. 12 13 14 T5 The resulting GeneScan plate record should appear as follows FA Sone ue Urea coe iniu colorc mca Bec Emp Deci Pix 15 O o ra k M nanpa 1 Campa TH Frege T Cumas Er OE Tort deicii MONFYG E N i UNA T NT LU TRA CL LI M ELO as X73 2S Tails mum Um mr Wiel TEREPE COAL ee ee RSEN HODSOE ROO a ON ECNEES MEE RCM ECH NES DUM NECS NECNON OE ENG RCM EC OMEN ZUM M ee INCENLNUR IM NEUEM N E LCNEIOE EE CUR EUN CX NOS NEC A LN ICUN ZUR DN EE IH RON NR DEC NOGCDUNUMN NCINCIESCTUUSE NUN ISSN DEC ON EN IEEE ISUN IC LM DGDUEM ZEROIUE E DEC SNCS ONG ACIE NR DE DUNS TCR IN HN SCSCNC I DUNT DUM IE E DR DUM DN DUCE DCN DOR IN ncm Click OK This new plate record will appear in the pending plate records table on the plate setup page of the collection software Place samples in instrument and close the instrument doors In the pending plate records table click once on the name of the plate record you just created Once the plate record is highlighted click the plate graphic that corresponds to the plate on the autosampler that contains your amplified samples When the plate record is linked to the plate the plate graphic will change from yellow to green the plate record moves from the pending plate records table to the linked plate records table and the Run Instrument button becomes enabled Click the Run Instrument button on the toolbar to start the sample run Monitor electrophores
6. Fax 608 277 2516 www promega com Printed in USA Part TMD009 Revised 6 07 Page 17 Promega Q Acrylamide is a neurotoxin Always wear double gloves and safety glasses when handling Note The gel can be stored overnight if a paper towel saturated with deionized water and plastic wrap are placed around the top and bot tom to prevent the gel from drying out crystal lization of the urea will destroy the gel VIII A Polyacrylamide Gel Preparation Acrylamide Long Ranger gel solution is a neurotoxin and suspected cancer agent avoid inhalation and contact with skin Read the warning label and take the necessary precautions Always wear gloves and safety glasses when working with acrylamide solutions The following protocol is for preparation of a 36cm denaturing polyacrylamide gel for use with the ABI PRISM 377 DNA sequencer Low fluorescence glass plates are recommended and can be obtained from the instrument manufacturer 1 Thoroughly clean the glass plates with hot water and a 1 Liqui Nox solution Rinse extremely well using deionized water Allow the glass plates to air dry in a dust free environment Assemble the glass plates by placing 0 2mm side gel spacers between the front and rear glass plates Hold the plates together using binder clamps 4 clamps on each side Place the assembly horizontally on a test tube rack or similar support Prepare a 5 Long Ranger acrylamide gel total of 50ml
7. 100RFU TMR labeled peaks might be observed at approximately 122 and 156 bases Create the desired table by selecting the PowerTable 310 for ABI PRISM9 310 or 3100 Genetic Analyzer data or PowerTable 377 for ABI PRISM 377 DNA Sequencer data Two additional table formats are available as macros Make Allele Table or Make CODIS Table You can customize these tables to fit the needs of your laboratory IX C Controls 1 Observe the lanes containing the negative controls They should be devoid of amplification products Observe the lanes containing the K562 DNA positive controls Amplified sample peak heights of less than 2 000RFU are ideal Compare the K562 DNA allelic repeat sizes with the locus specific allelic ladder The expected K562 DNA allele sizes for each locus are listed in Table 3 Section II Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Telephone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Revised 6 07 Part TMD009 Page 23 IX D Results Figure 1 shows typical results achieved using the PowerPlex 1 2 System and the ABI PRISM 310 Genetic Analyzer Wa ere odo ime Io Ma D e o c5 3 8 ie m ee Drs ra I1 717 LIO LET pu En A AR a J L iX pid EIEEREdE ka REESE Figure 1 Electrophoretic profile of a single DNA sample amplified using the PowerPlex 1 2 System
8. 5 or higher set macro displays an imported the preferences under edit to empty plot window import the blue green yellow and red colors Many off ladder Migration of samples Use a different injection of the peaks in the might change slightly over allelic ladder to determine sizes in samples using the the course of a CE run the PowerTyper 1 2 Macro Do ABI PRISM 310 with many samples This not use the first injection on a new or 3100 Genetic might be due to changes column for the ladder sample Analyzer in temperature or in the CE column over time The base pair size of the Confirm that the internal lane alleles is incorrect due to standard fragments are assigned incorrect fragment sizes correctly Reanalyze the sample assigned to the internal using GeneScan software and lane standard redefine the internal lane standard fragments Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Telephone 608 274 4330 Fax 608 277 2516 www promega com Part TMD009 Printed in USA Page 28 Revised 6 07 Promega Symptoms Possible Causes Comments F ii l Error message The peak heights for The allelic ladder categories are em caine e addressed here please Could not complete one or more of the allelic defined as having a minimum contact your local the Run Macro ladder sample files are peak height of 2OORFU If the Promega Branch Office command because below 200RFU ladder a
9. PIOGLUCISuternor ventes ibn rRUnb evipXtoE uf estesa nM uar np Rua ori r Mr b on RnNE NE dVbI 33 Description STR short tandem repeat loci consist of short repetitive sequence elements 3 to 7 base pairs in length 1 4 These repeats are well distributed throughout the human genome and are a rich source of highly polymorphic markers which can be detected using the polymerase chain reaction 5 8 Alleles of STR loci are differen tiated by the number of copies of the repeat sequence contained within the amplified region and are distinguished from one another using radioactive silver stain or fluorescence detection following electrophoretic separation The PowerPlex 1 2 System a b c allows the coamplification and two color detection of nine loci eight STR loci and Amelogenin The system contains all the component STR loci of two GenePrint quadriplex systems the GammaSTR System D16S539 D7S820 D13S317 and D5S818 and the GenePrint Fluorescent STR Multiplex CSF1PO TPOX THO1 and vWA CTTv multiplex as well as the GenePrint Fluorescent Sex Identification System Amelogenin TMR One of the two primers for each locus in the GammaSTR multiplex is labeled with fluorescein FL one primer is specific for each locus in the CTTv multiplex and one primer for Amelogenin is labeled with carboxy tetramethylrhodamine TMR All nine loci are amplified simultaneously in a single tube and analyzed in a single gel lane The PowerP
10. are not within the linear detection of the instru ment the ratio of stutter peaks to real allele peaks increases and allele designations become difficult to interpret The balance of the peak heights might also appear less uniform e There might be variation between instruments regarding the relative fluorescence units detected using the same sample Furthermore different instruments vary in the relative efficiency of color detection affecting the dye color to dye color balance VIII Detection of Amplified Fragments Using the ABI PRISM 377 DNA Sequencer Materials to Be Supplied by the User Solution compositions are provided in Section XII A e dry heating block water bath or e aerosol resistant pipette tips thermal cycler Section XII B e Long Ranger gel solution e Qgel loading pipette tips BioWhittaker Molecular e 36cm front and rear glass plates Applications Cat 50611 or Long e 36cm gel spacers 0 2mm thick Ranger Singel pack for ABI e 36 well sharkstooth comb or 34 well sequencers 377 36cm BioWhittaker squaretooth comb 0 2mm thick Molecular Applications Cat 50691 clamps e g large office binder e 10 ammonium persulfate clamps Cat V3131 e Liqui Nox9 detergent e TEMED e crushed ice e Urea Cat V3171 e TBE 10X buffer e Nalgene tissue culture filter 0 2 micron Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Telephone 608 274 4330
11. the thermal cycling proto problems cols in Section V B We have not tested other reaction tubes or thermal cyclers Calibration of the thermal cycler heating block might be required Primer concentration Use the recommended primer too low concentration Mix well before use Insufficient mixing of Vortex all reagents and cocktails reagents before use Samples not denatured Make certain the samples are heat before loading denatured and cooled on ice immediately prior to loading the gel or capillary Poor capillary electro Re inject the sample Check the phoresis injection syringe for leakage Check the Fluorescent Ladder laser power CXR also affected Poor quality formamide Ensure that high quality forma used mide is used when running sam ples on the ABI PRISM 310 and 3100 Genetic Analyzers The con ductivity of the deionized for mamide should be lt 100uUS cm For questions not addressed here please contact your local Promega Branch Office or Distributor Contact information available at www promega com E mail techserv promega com Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Telephone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Revised 6 07 Part TMD009 Page 25 Promega X A Amplification and Fragment Detection continued Symptoms Possible Causes Comments Extra peaks visible Contamination with C
12. Aerosol Resistant Tips Size Product Volume tips pack Cat ART 10 Ultramicro Pipet Tip 0 5 10ul 960 DY1051 ART 20E Ultramicro Pipet Tip 0 5 20ul 960 DY1061 ART 20P Pipet Tip 20ul 960 DY1071 ART GEL Gel Loading Pipet Tip 100yl 960 DY1081 ART 100 Pipet Tip 100yl 960 DY1101 ART 100E Pipet Tip 100ul 960 DY1111 ART 200 Pipet Tip 200yl 960 DY1121 ART 1000E Pipet Tip 1 000uI 800 DY1131 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Telephone 608 274 4330 Fax 608 277 2516 www promega com Part TMD009 Printed in USA Page 34 Revised 6 07 STR loci are the subject of U S Pat No RE 37 984 German Pat No DE 38 34 636 C2 and other patents issued to the Max Planck Gesellschaft zur F rderung der Wissenschaften e V Germany The development and use of STR loci are covered by U S Pat No 5 364 759 Australian Pat No 670231 and other pending patents assigned to Baylor College of Medicine Houston Texas Patents for the foundational PCR process European Pat Nos 201 184 and 200 362 expired on March 28 2006 In the U S the patents cover ing the foundational PCR process expired on March 29 2005 bD U S Pat Nos 5 843 660 and 6 221 598 Australian Pat No 724531 Canadian Pat No 2 118 048 Korean Pat No 290332 Singapore Pat No 57050 and Japanese Pat No 3602142 have been issued to Promega Corporation for multiplex amplification of STR loci Oth
13. Detection sees 21 G Reuse of Glass Plates erii mtis cet cue otii e soto atico ees ina o ceto 21 IX Data Analysis cisseutuieri ib vais ee uta bands MED oardan boas sania DRE RN DENIED Pad E AUG EE 21 A PowerTyper 1 2 Macro ss asscccsingeotcrtionnotadinsdaatnnaaysanmbinas Simedeunictslenaedestensccunasdasies 21 B ANAY eTm M 22 C Soi A m 23 De PO 0 m H H 24 X Iroubleshoollng dup isses uonb c ak vevas pO bv Svs genda e2 ss GuDVM E Sau dc mUs iE VL raS IG rues V RESUE 25 A Amplification and Fragment Detection ccccceccceseseeeeeeeeeeeeeeeeneeeeesaeeeeeas 25 B PowerTyper 1 2 Macro scessecisor bsumbdssiidsumr2d36da Ditu Stc bonu SES CU RUE Md tweamtduadeusaccn us 28 Xl Referentes cases sce umen mpm ewe ats gece eee scat 30 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Telephone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD009 Revised 6 07 Page 1 XII Note Information on other Promega fluores cent STR systems and detection of amplified STH fragments using silver staining 9 is available on the Internet at www promega com or upon request from Promega APREN OD d 32 A Composition of Buffers and Solutions eeeeeseeeeeeeeeereeenne 32 D Related
14. Formamide with a conductivity gt 100US cm might contain ions that compete with DNA during injection This results in lower peak heights and reduced sensitivity A longer injection time might not increase the signal VI A Matrix Standardization Proper generation of a matrix file is critical to evaluate multicolor systems with the ABI PRISM 310 Genetic Analyzer A matrix must be generated for each individual instrument The PowerPlex Matrix Standards 310 377 Cat DG3640 is required for matrix standardization for the ABI PRISM 310 Genetic Analyzer For best results the PowerPlex Matrix Standards 3100 Custom Cat X3121 should not be used to generate a matrix on the ABI PRISM 310 Genetic Analyzer Regardless of which instrument or matrix standards are used use only one JOE matrix fragment i e JOE Matrix A or JOE Matrix B when generating a matrix For protocols and additional information on matrix standardization see the PowerPlex Matrix Standards 310 377 Technical Bulletin TBDO18 which is supplied with Cat DG3640 This manual is available upon request from Promega or at www promega com tbs VI B Instrument Preparation 1 Refer to the ABI PRISM 310 Genetic Analyzer User s Manual for instruc tions on cleaning the pump block installing the capillary calibrating the autosampler and adding polymer to the syringe Open the ABI PRISM 310 data collection software Prepare a GeneScan sample sheet as des
15. HUMAMEL NA Note Amelogenin is and Y Human Y chromosomal gene not an STR but displays LLL forAmelogenindike protein a 212 base X specific Di6S589 FL 16gd qer NA 1 AGAT an D 880 FL 17q 1 1 1 NA 1 1 AAT r D135317 FL 18q22 d3 3 NA AGA D55818 FL 5g2i q31 M AGA CSF1PO TMR 5q33 3 34 HUMCSF1PO Human c fms proto AGAT oncogene for CSF 1 receptor gene TPOX TMR 2p23 2pter HUMTPOX AATG Human thyroid peroxidase gene THO1 TMR 11p15 5 HUMTHO 1 Human AATG tyrosine hydroxylase gene vWA TMR 12p12 pter HUMVWFA31 Human AGAT von Willebrand factor gene 1Repeat sequences represent all four possible permutations e g AGAT is used for AGAT GATA ATAG or TAGA The first alphabetic representation of the repeat e g AGAT is employed according to Edwards et al 2 TMR carboxy tetramethylrhodamine FL fluorescein NA not applicable Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Telephone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD009 Revised 6 07 Page 3 Note Information on strains 9947A 9948 and K562 is available online at locus umdnj edu nigms Strain K562 is available from the American Type Culture Collection www atcc org Manassas VA Table 2 The PowerPlex 1 2 System Allelic Ladder Information Size Range of Repeat Numbers Alle
16. PowerPlex 1 2 System Promega INSTRUCTIONS FOR USE OF PRODUCT DC6101 PLEASE DISCARD PREVIOUS VERSIONS All technical literature is available on the Internet at www promega com Please visit the web site to verify that you are using the most current version of this Technical Manual MES 1p es i ene Cee eae ee ee eee 2 k RR UNG re RN 2 A Advantages OF STR Typing siicccniiensissvcetuarncnctevadedinecdontnbuaniuseassnentvuasiiwadeucaesueotis 2 B Advantages of Using the Loci in the PowerPlex 1 2 System 9 SE doi don cedunt 1 ERU 4 D The Fluorescent Ladder CXR 60 400 Bases eese 6 Es Warnings and Precautions Jasruessemcundvapzserbaumadidur uice nt ria eade EEEa u EIRAN RU MUR EU RUE 6 Ill Product Components and Storage Conditions 7 IV DNA Extraction and Quantification Methods sseseuusssse 7 V Protocols for DNA Amplification Using the PowerPlex 1 2 System 8 A Amplification Sel uuccesidenii icone iin ana ux ncs Duci xuca dies Dess icum Deae bu RUE E Des ibn r de 8 B Amplification Thermal Cycling sicititeccaratatsmeesuntancenracerscactintiaccerinntenceciea cass 10 C Agarose Gel Electrophoresis of Amplification Products Optional 11 VI Detection of Amplified Fragments Using the ABI PRISM 310 Genetic Analyze
17. Quantitation System Cat DC1010 has been developed to work with the PowerPlex Systems 31 See Section XII B for ordering information Both the DNA IQ System and AluQuant Human DNA Quantitation System have been fully automated on the Beckman Coulter Biomek 2000 Laboratory Automation Workstation 32 For information on automation of laboratory processes on Beckman Coulter or other workstations contact your local Promega Branch Office or Distributor contact information available at www promega com or e mail techserv promega com Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Telephone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD009 Revised 6 07 Page 7 Q Meticulous care must be taken to ensure successful amplification A guide to amplification troubleshooting is provided in Section X A Note If using the GeneAmp PCR System 2400 9600 or 9700 ther mal cyclers use 0 2ml thin walled MicroAmp reaction tubes For the Perkin Elmer Model 480 the standard 0 5ml GeneAmp reaction tubes are recommended Protocols for DNA Amplification Using the PowerPlex 1 2 System Materials to Be Supplied by the User e thermal cycler model 480 or GeneAmp system 2400 9600 or 9700 Applied Biosystems microcentrifuge 0 5ml or 0 2ml thin walled microcentrifuge tubes Applied Biosystems 1 5ml amber colored microcentrifuge tubes Fis
18. STR loci PowerPlex 1 2 System 0 99990 0 99981 0 99974 combined with FFFL multiplex 12 STR loci Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Telephone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD009 Revised 6 07 Page 5 l1 D The Fluorescent Ladder CXR 60 400 Bases The Fluorescent Ladder CXR 60 400 Bases is an internal lane standard that contains 16 evenly spaced DNA fragments of 60 80 100 120 140 160 180 200 225 250 275 300 325 350 375 and 400 bases in length Each fragment is labeled with carboxy X rhodamine CXR and can be detected separately as a third color in the presence of PowerPlex 1 2 System amplified material using the Applied Biosystems DNA sequencers This ladder can be used as an internal size standard in each lane to increase precision in analyses when using the PowerPlex 1 2 System A protocol for preparation and use of this internal lane standard is provided in Sections VI C VII B and VIII E Il E Warnings and Precautions The application of PCR based typing for forensic or paternity casework requires validation studies and quality control measures that are not contained in this manual 28 29 The quality of the purified DNA sample as well as small changes in buffers ionic strength primer concentrations choice of thermal cycler and thermal cycling conditions can affect the success of PCR We s
19. Telephone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD009 Revised 6 07 Page 31 XII Appendix XII A Composition of Buffers and Solutions 10 ammonium persulfate Add 0 05g of ammonium persulfate to 500yl of deionized water Blue Dextran Loading Solution 88 25 formamide 15mg ml blue dextran 4 1mM EDTA pH 8 0 0 5M EDTA pH 8 0 stock 186 19 NasEDTA 2H20 Add EDTA to 800ml of deionized water with vigorous stirring Adjust the pH to 8 0 with NaOH about 20g of NaOH pellets Dispense into aliquots and sterilize by autoclaving ethidium bromide solution 10mg ml 1g ethidium bromide Dissolve the ethidium bromide in 100ml of deionized water Wrap in aluminum foil or transfer the solution to a dark bottle and store at room temperature Caution Ethidium bromide is a powerful mutagen Gloves should be worn when working with the dye and a mask should be worn when weighing it out Gold ST R 10X Buffer 500mM KCI 100mM Tris HCl pH 8 3 at 25 C 15mM MgClo 19e Triton X 100 2mM each dNTP 1 6mg ml BSA TAE 50X buffer pH 7 2 242g Tris base 57 1ml glacial acetic acid 100ml 0 5M EDTA stock Add the Tris base and EDTA stock to 500ml of deionized water Add the glacial acetic acid Bring to 1 liter with deionized water TBE 10X buffer 107 8g Tris base 7 44g Na2EDTA e 2H20 55g boric acid Dissolve the Tris base and EDTA in 800ml of deionized water Slowly add the bor
20. and detected with the ABI PRISM 310 Genetic Analyzer The fluorescein labeled loci D168539 D7S820 D13S317 and D58818 are displayed in the top panel The TMR labeled loci CSF1PO TPOX Amelogenin THO1 and vWA are displayed in the middle panel The Fluorescent Ladder CXR 600 400 Bases is shown in the bottom panel and is used as the internal lane standard Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Telephone 608 274 4330 Fax 608 277 2516 www promega com Part TMD009 Printed in USA Page 24 Revised 6 07 X Troubleshooting X A Amplification and Fragment Detection Symptoms Possible Causes Comments Faint or no allele Impure template DNA Because of the small amount of peaks template used this is rarely a problem Depending on the DNA extraction procedure used inhibitors might be present in the DNA sample Insufficient template Use the recommended amount of DNA template DNA Insufficient enzyme Use the recommended amount of activity AmpliTag Gold DNA polymerase Check the expiration date on the tube label Incorrect amplification Confirm the amplification program program High salt concentration The DNA volume should not be or altered pH more than 20 of the total reac tion volume Carryover from the DNA sample of K Na Mg or EDTA can have a deleterious effect on PCR A change in pH might also affect PCR Thermal cycler or tube Review
21. arison between the allelic ladder and amplified samples of the same locus allows rapid and precise assignment of alleles Results obtained using the PowerPlex 1 2 System can be recorded in a digitized format allowing direct comparison with stored databases Population analyses do not require the use of arbitrarily defined fixed bins for population data 12 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Telephone 608 274 4330 Fax 608 277 2516 www promega com Part TMD009 Page 2 Printed in USA Revised 6 07 II B Advantages of Using the Loci in the PowerPlex 1 2 System The loci included in the PowerPlex 1 2 System Table 1 have a high degree of heterozygosity and display a minimum of artifacts Each STR locus also displays alleles within a limited size range Table 2 This allows simultaneous detection of several loci without overlap of alleles across loci The sex identification locus Amelogenin is also included in the system Table 3 lists the PowerPlex 1 2 System allele determinations for commonly available standard DNA templates STR loci and primers were carefully selected to avoid or minimize artifacts including those associated with Jaq DNA polymerase such as repeat slippage and terminal nucleotide addition as well as genetic artifacts called microvariant alleles Repeat slippage 13 14 known as n 4 bands stutter or shadow bands is du
22. by combining the ingredients listed in Table 9 Stir the solution until the urea has dissolved Table 9 Preparation of a 5 Long Ranger Polyacrylamide Gel Component 5 Gel Final Concentration urea 18g 6M deionized water 26ml 10X TBE buffer 5ml 1X 5096 Long Ranger gel solution 5ml 596 total volume 50ml Note Long Ranger Singel Packs can be used Filter the acrylamide solution through a 0 2 micron filter e g Nalgene tissue culture filter and degas for an additional 5 minutes Add 35ul of TEMED and 250ul of fresh 10 ammonium persulfate to the 50ml of acrylamide solution and mix gently Using a disposable 30cc syringe pour the gel by starting at the well end of the plates and carefully injecting the acrylamide between the horizontal glass plates Allow the solution to fill the top width of the plates While main taining a constant flow of solution gently tap the glass plates to assist the movement of solution to the bottom of the plates and to prevent the forma tion of bubbles Insert a 36 well sharkstooth comb or 34 well squaretooth comb between the glass plates Sharkstooth combs with 64 or 96 wells can also be used Secure the comb with 3 evenly spaced clamps Keep the remaining acrylamide solution as a polymerization control Allow polymerization to proceed for at least 2 hours Check the polymerization control to be sure that polymerization has occurred Promega Corporation 2800 Woods Hollow Roa
23. c Ladder Mix Cover wells with appropriate septa Note Instrument detection limits vary therefore injection time or the amount of product mixed with loading cocktail might need to be increased or decreased If the peak heights are too high 22 000RFU the samples can be diluted in Gold ST R 1X Buffer before mixing with loading cocktail This can result in uneven allele peak heights across loci For best results use less DNA template in the amplification reactions or reduce the number of cycles in the amplification program by 2 4 cycles Denature samples at 95 C for 3 minutes then immediately chill on crushed ice for 3 minutes Denature the samples just prior to loading the ABI PRISM 3100 Genetic Analyzer VII C Instrument Preparation 1 Refer to the ABI PRISM 3100 Genetic Analyzer User s Manual for instruc tions on cleaning the pump blocks installing the capillary array performing a spatial calibration and adding polymer to the reserve syringe Open the ABI PRISM 3100 data collection software Open a new plate record Name the plate and select GeneScan Select the plate size 96 well or 384 well Click Finish Complete the plate record spreadsheet for the wells you have loaded Enter appropriate information into the Sample Name and Color Info columns For Allelic Ladder samples insert the word ladder into the Color Info column for the blue yellow and green dye colors This information must be ent
24. cipitate might form in the Gold ST R Buffer If this occurs warm the buffer briefly at 37 C then vortex until it is in solution 2 Determine the number of reactions to be set up This should include positive and negative control reactions Add 1 or 2 reactions to this number to com pensate for pipetting error While this approach does waste a small amount of each reagent it ensures that you will have enough PCR master mix for all samples It also ensures that each reaction contains the same master mix 3 Place one clean 0 2ml or 0 5ml microcentrifuge tube for each reaction into a rack and label appropriately 4 Calculate the required amount of each component of the PCR master mix Table 8 Multiply the volume ul per sample by the total number of reac tions from Step 2 to obtain the final volume ul 5 Inthe order listed in Table 8 add the final volume of each reagent into a sterile 1 5ml amber colored tube Mix gently Place on crushed ice if using Taq DNA polymerase Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Telephone 608 274 4330 Fax 608 277 2516 www promega com Part TMD009 Page 8 Printed in USA Revised 6 07 Promega Table 8 Preparation of Master Mix for the PowerPlex 1 2 System PCR Master Volume Per Numberof Final Mix Component Sample Reactions Volume ul Nuclease Free Water m5 Gold ST R 10X Buffer 2 5yl PowerPlex 1 2 10X Pri
25. cribed in the AB PRISM 310 Genetic Analyzer User s Manual Enter the appropriate sample information in the sample info column For the red dye color insert ILS i e internal lane standard to represent the Fluorescent Ladder CXR 60 400 Bases For rows containing the PowerPlex 1 2 Allelic Ladder Mix insert Ladder in the sample info column for the blue dye color and yellow dye color This information must be entered to successfully analyze your data using the PowerTyper 1 2 Macro available online at www promega com geneticidtools or upon request from Promega Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Telephone 608 274 4330 Fax 608 277 2516 www promega com Part TMD009 Page 12 Printed in USA Revised 6 07 4 Create a new GeneScan injection list Select the appropriate sample sheet by using the pull down menu 5 Select the GS STR POPA 1ml A Module using the pull down menu Change the injection time to 2 seconds and keep the default settings for the remaining parameters as shown below Inj Secs 2 Inj kV 15 0 Run kV 15 0 Run C 60 Run Time 24 Select the appropriate matrix file Section VII A To analyze the data automatically select the auto analyze checkbox and the appropriate analysis parameters and size standard Refer to the AB PRISM 310 Genetic Analyzer User s Manual for specific information on these opti
26. cycles or long term storage at 4 C can cause a breakdown of the formamide Formamide with a conductivity gt 100uUS cm might contain ions that compete with DNA during injection This results in lower peak heights and reduced sensitivity A longer injection time might not increase the signal VII A Spectral Calibration Proper generation of a matrix file is critical to evaluate multicolor systems with the ABI PRISM 3100 Genetic Analyzer A matrix must be generated for each individual instrument The PowerPlex Matrix Standards 3100 Custom Cat X3121 is required for spectral calibration on the ABI PRISM 3100 Genetic Analyzer The PowerPlex Matrix Standards 310 377 Cat DG3640 cannot be used for spectral calibra tion on this instrument Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Telephone 608 274 4330 Fax 608 277 2516 www promega com Part TMD009 Page 14 Printed in USA Revised 6 07 VII B Sample Preparation The Fluorescent Ladder CXR 60 400 Bases Cat DG6221 is included with the PowerPlex 1 2 System as the internal lane standard 1 Prepare a loading cocktail by combining and mixing the internal lane standard and Hi Di formamide as follows 1ul ILS x injections 9ul Hi Di formamide x injections Pipet 10ul of formamide internal lane standard mix into each well Add 1ul of amplified sample or 1ul of the Alleli
27. d Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Telephone 608 274 4330 Fax 608 277 2516 www promega com Part TMD009 Page 18 Printed in USA Revised 6 07 VIII B Matrix Standardization Proper generation of a matrix file is critical to evaluate multicolor systems with the ABI PRISM 377 DNA Sequencer A matrix must be generated for each individual instrument The PowerPlex Matrix Standards 310 377 Cat DG3640 is required for matrix standardization for the ABI PRISM 377 DNA Sequencer For best results the PowerPlex Matrix Standards 3100 Custom Cat X3121 should not be used to generate a matrix on the ABI PRISM 377 DNA Sequencer For protocols and additional information on matrix standardization see the PowerPlex Matrix Standards 310 377 Technical Bulletin TBDO18 which is supplied with Cat DG3640 This manual is available upon request from Promega or at www promega com tbs VIII C Instrument Preparation 1 Open the ABI PRISM 377 data collection software 2 Prepare a sample sheet as described in the GeneScan Analysis Software User s Manual Enter the appropriate sample information in the sample info column For the red dye color insert ILS i e internal lane standard to represent the Fluorescent Ladder CXR 600 400 Bases For rows containing the PowerPlex 1 2 Allelic Ladder Mix insert Ladder for the blue dye color and yellow dye color This information must be
28. d by pending or issued patents or may have certain limitations Please visit our Web site for more information All prices and specifications are subject to change without prior notice Product claims are subject to change Please contact Promega Technical Services or access the Promega online catalog for the most up to date information on Promega products Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Telephone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD009 Revised 6 07 Page 35 Printed in USA Revised 6 07 Part TMD009 PowerPlex 1 2 system 1998 2007 Promega Corporation All Rights Reserved Promega Corporation C 2800 Woods Hollow Road Madison WI 53711 5399 USA e Telephone 608 274 4330 Fax 608 271 2516 Prom TAREA CNET AF9TMDOO9 0607TMDOO9
29. d run a recommended protocol The preferred protocols for use with the GeneAmp PCR system 9700 the GeneAmp PCR system 9600 the GeneAmp PCR system 2400 and the Perkin Elmer model 480 thermal cyclers are provided below 3 After completion of the thermal cycling protocol store the samples at 20 C in a light protected box Protocol for the Perkin Elmer GeneAmp PCR System 9700 Thermal Cycler Ramp Speed 9600 95 C for 11 minutes then 96 C for 1 minute then ramp 100 to 94 C for 30 seconds ramp 29 to 60 C for 30 seconds ramp 23 to 70 C for 45 seconds for 10 cycles then ramp 100 to 90 C for 30 seconds ramp 29 to 60 C for 30 seconds ramp 23 to 70 C for 45 seconds for 20 cycles then 60 C for 30 minutes 4 C soak Protocol for the Perkin Elmer GeneAmp PCR System 9600 Thermal Cycler 95 C for 11 minutes then 96 C for 1 minute then 94 C for 30 seconds ramp 68 seconds to 60 C hold for 30 seconds ramp 50 seconds to 70 C hold for 45 seconds for 10 cycles then 90 C for 30 seconds ramp 60 seconds to 60 C hold for 30 seconds ramp 50 seconds to 70 C hold for 45 seconds for 20 cycles then 60 C for 30 minutes 4 C soak Protocol for the Perkin Elmer GeneAmp PCR System 2400 Thermal Cycler 95 C for 11 minutes then 96 C for 1 minute then ramp 100 to 94 C for 30 seconds ramp 100 to 60 C for 30 seconds ramp 23 to 70 C for 45 seconds for 10 cycles then
30. e to the loss of a repeat unit during DNA amplification somatic variation within the DNA in sample material or both The amount of this artifact observed depends primarily on the locus and the DNA sequence being repli cated The loci chosen exhibit little or no repeat slippage The vWA locus is an exception revealing as much as 10 stutter This locus has been included pri marily for its popularity in the forensic DNA testing community Terminal nucleotide addition 15 16 occurs when Tag DNA polymerase adds a nucleotide generally adenine to the ends of amplified DNA fragments in a template independent manner Thus an artifact band one base shorter than expected i e missing the terminal addition is sometimes seen We have modi fied primer sequences and added a final extension step of 60 C for 30 minutes 17 to the amplification protocol to provide conditions of essentially full terminal nucleotide addition The presence of microvariant alleles complicates separation interpretation and assignment of alleles There appears to be a correlation between a high degree of polymorphism a tendency for microvariants and increased mutation rate 18 19 Therefore we have selected loci with moderately high polymorphism and minimal occurrence of artifacts Table 1 The PowerPlex 1 2 System Locus Specific Information STR Chromosomal GenBank Locus Repeat Sequence Locus Label Location and Definition SS Amelogenin TMR Xp22 10 22 3
31. entered to successfully analyze your data using the PowerTyper M 1 2 Macro 3 Create a new GeneScan run and use the following settings Plate Check Module Plate Check A PreRun Module PR GS 36A 2400 Run Module GS 36A 2400 Collect Time 2 5 hours Well to Read Distance 36cm 4 Select the appropriate sample sheet and comb selection by using the pull down menus 5 Select the appropriate gel matrix file Section VIII B VIII D Gel Prerun 1 Remove the clamps from the polymerized acrylamide gel If necessary clean any excess acrylamide from the glass plates with paper towels saturated with deionized water 2 Shave any excess polyacrylamide away from the comb and remove the comb If using a sharkstooth comb carefully insert the sharkstooth comb teeth into the gel approximately 1 2mm 3 Position the gel glass plate unit in the 377 cassette Secure the cassette in the instrument and perform a plate check as recom mended in the ABI PRISM 377 DNA Sequencer User s Manual f the horizontal line graph is not flat remove the cassette clean the plate surface and repeat the plate check 5 Add TBE 1X buffer to the top and bottom buffer chambers of the instrument Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Telephone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD009 Revised 6 07 Page 19 8 Using a 30cc syringe filled with bu
32. er patents are pending c The purchase of this product does not convey a license to use AmpliTaq Gold DNA polymerase You should purchase AmpliTaq Gold DNA polymerase licensed for the forensic and human identity field directly from your authorized enzyme supplier 1998 2007 Promega Corporation All Rights Reserved AluQuant GammaSTR GenePrint and PowerPlex are registered trademarks of Promega Corporation DNA IQ and PowerTyper are trademarks of Promega Corporation ABI PRISM GeneScan Genotyper and MicroAmp are registered trademarks of Applera Corporation AmpliTaq AmpliTaq Gold and GeneAmp are registered trademarks of Roche Molecular Systems Inc ART is a registered trademark of Molecular Bio Products Inc Biomek is a registered trademark of Beckman Coulter Inc FTA is a registered trademark of Flinders Technologies Pty Ltd and is licensed to Whatman GenBank is a registered trademark of U S Dept of Health and Human Services Hi Di POP 4 and POP 6 are trademarks of Applera Corporation Liqui Nox is a registered trademark of Alconox Inc Long Ranger and Long Ranger Singel are registered trademarks of Cambrex Corporation Macintosh is a registered trademark of Apple Computer Inc Nalgene is a registered trademark of Nalge Nunc International Triton is a registered trademark of Union Carbide Chemicals and Plastics Technology Corporation Windows and Windows NT are registered trademarks of Microsoft Corporation Products may be covere
33. ered to successfully analyze data with the PowerTyper 1 2 Macro In the BioLIMS Project column select 3100 Project1 from the pull down menu In the Dye Set column select Z from the pull down menu In the Run Module 1 column select GeneScan36_POP4DefaultModule from the pull down menu To collect the data without autoanalyzing select No Selection in the Analysis Module 1 column Analysis parameters can be applied after data collection and during data analysis using the GeneScan analysis software To analyze the data during data collection an appropriate analysis module must be selected in the Analysis Module 1 column Refer to the AB PRISM 3100 Genetic Analyzer User s Manual for specific instructions on creating analysis modules Note The volume of internal lane standard used in the loading cock tail can also be increased or decreased to adjust the intensity of the size standard peak heights Note Brief centrifugation of prepared samples will remove bubbles that might affect analysis Note If less signal inten sity is desired injection time can be decreased to 5 seconds in the run module Use the Module Editor under Tools to change the run module Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Telephone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD009 Revised 6 07 Page 15 10 11
34. es in temperature or in the CE column over time Excessive amount of We recommend 1 2ng of DNA DNA template Amplification of DNA templates at greater than 2ng results in an imbalance in yields with the smaller loci showing more amplification product than the larger loci Use less DNA template or reduce the number of cycles in the amplification program by 2 4 cycles 10 18 or 10 16 cycling to improve locus to locus balance Results might be similar to use of excess amounts of DNA template Reduce the number of cycles in the amplification program by 2 4 cycles 10 18 or 10 16 cycling to improve locus to locus balance DNA template is degraded and the larger loci show diminished yield Insufficient template Use the recommended amount of DNA template DNA Poor quality formamide Ensure that high quality formamide used is used when running samples on the ABI PRISM 310 and 3100 Genetic Analyzers The conductivity of the deionized formamide should Use of FTA paper Degraded DNA sample be 100p S cm For questions not addressed here please contact your local Promega Branch Office or Distributor Contact information available at www promega com E mail techserv promega com Note Dilution of over amplified samples displays dropoff of larger loci Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Telephone 608 274 4330 Fax 608 277 2516 www p
35. f cycles in the ampli fication program by 2 4 cycles 10 18 or 10 16 cycling Pull up or bleedthrough Pull up can occur when peak heights are gt 2 000RFU or if a poor or incorrect matrix has been applied to the samples Increase the peak amplitude threshold i e 150 200RFU in the analysis parameters and reanalyze using the GeneScan software Generate a new matrix and apply to the samples Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Telephone 608 274 4330 Fax 608 277 2516 www promega com Part TMD009 Printed in USA Page 26 Revised 6 07 Symptoms Possible Causes Comments Allelic ladder is not running the same as the sample Imbalance of peak heights Allelic ladder and primer pair mix not Ensure that the allelic ladder is from the same kit as the primer compatible pair mix Poor quality formamide Ensure that high quality formamide used is used when running samples on the ABI PRISM 310 and 3100 Genetic Analyzers The conductivity of the deionized formamide should be lt 100uS cm Samples were diluted in the wrong buffer Use 1X Gold ST R Buffer to dilute samples Use a different injection of the allelic ladder to determine sizes in the PowerTyper 1 2 Macro Buffer incompatibility Migration of samples can change slightly over the course of a capillary electrophoresis CE run with many samples This might be due to chang
36. ferent instruments vary in the relative efficiency of color detection affecting the dye color to dye color balance VIII G Reuse of Glass Plates Note Soap and oil can build up on plates result Separate the glass plates and discard the gel Clean the glass plates with hot Ing in gel extrusion or hazy background Plates can be soaked in 2N HCI for 15 minutes then rinsed thoroughly water and a detergent such as 1 Liqui Nox9 detergent Rinse extremely well with deionized water and allow the plates to air dry Do not scrape the plates with abrasive materials during this process IX Data Analysis IX A PowerTyper 1 2 Macro Q The To facilitate analysis of the data generated with the PowerPlex 1 2 System we PowerTyper Macro is have created a file to allow automatic assignment of genotypes using the a Genotyper file and can be overwritten if save is used instead of Save as Genotyper software After samples have been amplified using the PowerPlex 1 2 System detected using the ABI PRISM 310 or 3100 Genetic Analyzer or 377 DNA sequencer and analyzed using the GeneScan analysis software the sample files can be imported into the Genotyper program and analyzed using the PowerTyper M 1 2 Macro The PowerTyper 1 2 Macro is provided on the PowerTyper Macros CD ROM Cat DG3470 available upon request from Promega PowerTyper files can also be downloaded from our web site at www promega com geneticidtool
37. ffer remove any air bubbles from the well area of the gel and place the lid on the upper buffer chamber Using a syringe with a bent 19 gauge needle remove any air bubbles from the bottom of the gel Attach the heating plate connect the water tubing attach all electrodes close the instrument door and select PreRun Allow the gel to prerun for 15 20 minutes or until the gel temperature is at least 40 C Open the status window to monitor the temperature of the gel Prepare the sample and allelic ladder samples during the gel prerun VIILE Sample Preparation and Loading The Fluorescent Ladder CXR 60 400 Bases is included in the PowerPlex 1 2 System as the internal lane standard ILS for three color detection and analysis of amplified samples With this approach only 2 3 lanes of the PowerPlex 1 2 Allelic Ladder Mix are required per gel 1 o You might need to optimize Step 9 for individual 8 9 instruments Loading volumes of 1 0 2 0ul Prepare a loading cocktail by combining and vortexing the Fluorescent Ladder CXR and Blue Dextran Loading Solution as follows 0 5ul Fluorescent Ladder x lanes 1 5ul Blue Dextran Loading Solution x lanes Vortex for 10 15 seconds Combine 2ul of the prepared loading cocktail and 0 5 1 ul of amplified sam ple Mix by pipetting Note Instrument detection limits vary therefore the amount of product mixed with loading cocktail might need to be
38. heat in a microwave oven to dissolve the agarose Add preheated 60 C deionized water to make up for any volume lost due to evaporation 2 Cool the agarose to 55 C before pouring into the gel tray Be sure that the gel tray is level Pour the agarose into the tray insert the gel comb and allow to set for 20 30 minutes 3 Prepare the samples by mixing 10ul of each amplified sample with 2 5ul of 5X loading solution 4 Prepare 1 liter of TAE 1X buffer for the electrophoresis running buffer Place the gel and tray in the electrophoresis gel box Pour enough running buffer into the tank to cover the gel to a depth of at least 0 65cm Gently remove the comb Load each sample mixed with 5X loading solution see Step 3 above 7 Set the voltage at 5 volts cm measured as the distance between the two electrodes Allow the gel to run for 2 hours 8 After electrophoresis stain the gel in TAE 1X buffer containing 0 5ug ml ethidium bromide Gently rock for 20 minutes at room temperature Remove the ethidium bromide solution and replace with deionized water Allow the gel to destain for 20 minutes 9 Photograph the gel using a UV transilluminator 302nm Note When analyzing the data do not be alarmed if you see extra bands in addition to the alleles DNA heteroduplexes can be expected when perform ing nondenaturing agarose gel electrophoresis The sole purpose of the agarose gel is to confirm the success of the PCR Promega Cor
39. her Cat 05 402 26 aerosol resistant pipette tips Section XII B e AmpliTaq Gold DNA polymerase Applied Biosystems e Nuclease Free Water Cat P1193 e Mineral Oil Cat DY1151 for use with the thermal cycler model 480 We routinely amplify 1ng of template DNA in a 25ul reaction volume using the protocols detailed below Amplification protocols for the Perkin Elmer model 480 and the GeneAmp PCR system 2400 9600 and 9700 thermal cyclers are provided The PowerPlex 1 2 System has been developed for amplification using AmpliTaq Gold DNA polymerase and Gold ST R 10X Buffer AmpliTag DNA polymerase can be used with the Gold ST R 10X Buffer and the PowerPlex 1 2 System but the balance of product yield among the loci might be less uniform than with the use of AmpliTaq Gold DNA polymerase V A Amplification Setup The use of gloves and aerosol resistant pipette tips is highly recommended to prevent cross contamination Keep all pre amplification and postamplification reagents in separate rooms Prepare amplification reactions in a room dedicated for reaction setup Use equipment and supplies dedicated for amplification setup 1 Thaw the Gold ST R 10X Buffer and PowerPlex 1 2 10X Primer Pair Mix Notes e tis very important to mix these reagents by vortexing for 5 10 seconds before each use Do not centrifuge the 10X Primer Pair Mix as this might cause the primers to be concentrated at the bottom of the tube e A pre
40. ic acid and monitor the pH until the desired pH of 8 3 is obtained Bring the volume to 1 liter with deionized water TE 4 buffer 10mM Tris HCI 0 1mM EDTA pH 7 5 2 21g Tris base 0 037g NasEDTA e 2H20 Dissolve Tris base and EDTA in 900ml of deionized water Adjust to pH 7 5 with HCI Increase volume to 1 liter with deionized water Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Telephone 608 274 4330 Fax 608 277 2516 www promega com Part TMD009 Printed in USA Page 32 Revised 6 07 Promega XII B Related Products GenePrint Fluorescent STR Multiplex Systems Product Size Cat PowerPlex 16 System 100 reactions DC6531 400 reactions DC6530 PowerPlex ES System 100 reactions DC6731 400 reactions DC6730 GammaSTR Multiplex Fluorescein 100 reactions DC6071 400 reactions DC6070 GenePrint Fluorescent STR Multiplex CSF 1 PO TPOX THO1 vWA Fluorescein CTTv Multiplex 100 reactions DC6301 400 reactions DC6300 GenePrint Fluorescent STR Multiplex F 13A01 FESFPS F13B LPL Fluorescein FFFL Multiplex 100 reactions DC6311 400 reactions DC6310 Not for Medical Diagnostic Use GenePrint9 Sex Identification Systems Product Size Cat GenePrint Fluorescent Sex Identification system Amelogenin Fluorescein 100 reactions DC5171 GenePrint Fluorescent Sex Identification system Amelogenin TMR 100 reactions DC6171 Not for Medical Diagnostic Use Alle
41. increased or decreased If the peak heights are too high i e greater than 2 000RFU the samples can be diluted in Gold ST R 1X Buffer before mixing with loading cocktail This might result in uneven allele peak heights across loci For best results use less DNA template in the amplification reactions or reduce the number of cycles in the amplification program by 2 4 cycles i e 10 18 or 10 16 cycling Combine 2ul of the prepared loading cocktail and 1ul of the PowerPlex 1 2 Allelic Ladder Mix vortex the Allelic Ladder Mix prior to pipetting Note To run the THO1 Allele 9 3 alone prepare a 1 4 dilution 1 part THO1 Allele 9 3 with 3 parts 1X STR Buffer and combine 1yl of this dilution with 2ul of loading cocktail To run the THO1 Allele 9 3 in combination with the PowerPlex 1 2 Allelic Ladder prepare a 1 3 mixture 1 part THO1 Allele 9 3 with 3 parts PowerPlex 1 2 Allelic Ladder then combine 1 yl of this mixture with 2ul of loading cocktail Briefly spin the samples in a microcentrifuge to bring the contents to the bottom of the tubes Denature the samples by heating at 95 C for 2 minutes and immediately chill on crushed ice Denature the samples just prior to loading the gel After the 15 to 20 minute prerun pause the instrument by clicking on the pause button By pausing the prerun the water will continue to circulate to keep the gel warm during the sample loading Use a 30cc syringe filled with buffer to flush
42. is by observing the run status array and capillary views windows in the collection software Each run 16 samples capillaries will take approximately 45 minutes VII D Sample Detection Analyze the data using the GeneScan analysis software Heview the raw data for one or more sample runs Highlight the sample file name then under the sample menu select raw data Move the cursor so the crosshair is on the baseline to the right of the large primer peak before the first internal lane standard peak red Use the X value number shown at the bottom left of the window for the start position in the analysis parameters The recommended analysis parameters are Analysis Range Start Defined in Step 2 Stop 10 000 Data Processing Baseline Checked Multicomponent Checked Smooth Options Light Peak Detection Peak Amplitude Thresholds B Y G R Min Peak Half Width 2pts Size Call Range Min 60 Max 600 Size Calling Method Local Southern Method Split Peak Correction None 1Smoothing options should be determined by individual laboratories Occasionally the separation control alleles and the THO1 alleles 9 3 and 10 will not be distinguished using heavy smoothing 2The peak amplitude thresholds are the minimum peak height that the software will call as a peak Values for the peak amplitude thresholds are usually 50 200RFU and should be deter mined by individual laboratories Promega Corporation 2800 Woods Hollow Road
43. l approach to analysis of polymorphic short tandem repeat loci Bio Techniques 20 266 76 25 Lins A M et al 1996 Multiplex sets for the amplification of polymorphic short tandem repeat loci silver stain and fluorescent detection Bio Techniques 20 882 9 26 Jones D A 1972 Blood samples Probability of discrimination J Forensic Sci Soc 12 355 9 27 Brenner C and Morris J W 1990 In Proceedings from the International Symposium on Human Identification 1989 Promega Corporation 21 53 28 Presley L A et al 1992 The implementation of the polymerase chain reaction PCR HLA DQ alpha typing by the FBI Laboratory In The Third International Symposium on Human Identification 1992 Promega Corporation 245 69 29 Hartmann J M et al 1991 Guidelines for a quality assurance program for DNA analysis Crime Laboratory Digest 18 44 75 30 Mandrekar P V Krenke B E and Tereba A 2001 DNA IQ The intelligent way to purify DNA Profiles in DNA 4 3 16 31 Mandrekar M N et al 2001 Development of a human DNA quantitation system Profiles in DNA 4 8 9 12 32 Greenspoon S and Ban J 2002 Robotic extraction of sexual assault samples using the Biomek 2000 and the DNA IQ System Profiles in DNA 5 1 3 5 Additional STR references can be found at www promega com geneticidentity Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526
44. le fragments with allelic ladders and internal lane standards Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Telephone 608 274 4330 Fax 608 277 2516 www promega com Part TMD009 Page 22 Printed in USA Revised 6 07 When using an internal size standard the calculated lengths of the allelic ladder components will differ from those listed in the table This is due to differences in migration resulting from sequence differences between the allelic ladder fragments and those of the internal size standard and is nota matter of concern Double click on the CTATv Ladders macro A plots window will open to display the yellow dye allelic ladders i e CTATv allelic ladders CSF1PO TPOX Amelogenin THO1 and vWA Confirm that the correct allele numbers were assigned to the allelic ladders Double click on the Display GammaSTR macro to display the blue dye for all sample injections lanes Scroll down to observe and or edit as necessary Double click on the Display CTATv macro to display the yellow dye for all sample injections lanes Scroll down to observe and or edit as necessary Notes If samples are overloaded a possible off ladder allele might be detected in the vWA allele size range approximately 19 20 bases smaller than the allele representing a homozygous sample When using the ABI PRISM 377 DNA Sequencer and samples are over loaded broad and small less than
45. lex 1 2 System is designed specifically for use with the ABI PRISM 310 Genetic Analyzer and is compatible with the ABI PRISM 377 DNA Sequencer and ABI PRISM 3100 Genetic Analyzer The PowerPlex 1 2 System provides all of the materials necessary for amplification of STR regions of purified genomic DNA with the exception of AmpliTaq Gold DNA polymerase This manual contains sepa rate protocols for use of the PowerPlex 1 2 System with the Perkin Elmer model 480 and GeneAmp PCR system 2400 9600 and 9700 thermal cyclers in addition to protocols for separation of amplified products and detection of separated material Protocols for operation of the fluorescence detecting instrumentation should be obtained from the manufacturer STR Typing II A Advantages of STR Typing STR typing is more tolerant of the use of degraded DNA templates than other typing methods because the amplification products are less than 500bp long much smaller than the material detected using AMP FLP 10 or VNTR 11 analysis STR typing is also amenable to a variety of rapid DNA purification techniques which are compatible with PCR but do not provide enough DNA of appropriate quality for Southern blot based analyses Promega STR amplification products are generally of discrete and separable lengths This allows the construction of allelic ladders containing fragments of the same lengths as several or all Known alleles for each locus Visual or instru ment based comp
46. lic Ladder Repeat Numbers of of Alleles Not STR Components 2 Allelic Ladder Present in Locus Label bases Components Allelic Ladder Amelogenin TMR 212 X 218 Y NA None D16S539 FL 264 304 5 8 15 None D7S820 FL 215 247 6 14 None D139317 FL 176 208 7 15 None D55818 FL 119 151 7 15 None CSF1PO TMR 291 327 6 15 None TPOX TMR 224 252 6 13 None THO1 TMR 179 203 5 11 8 33 9 33 vWA TMR 127 167 11 13 21 None 1Lengths of each allele in the allelic ladders have been confirmed by sequence analyses 2When using an internal lane standard such as the Fluorescent Ladder CXR 60 400 Bases calculated sizes of allelic ladder components might differ from those listed 3The THO1 allele 9 3 is quite common while allele 8 3 is extremely rare Other extremely rare variants have been reported at the THO1 locus Table 3 The PowerPlex 1 2 System Allele Determinations in Commonly Available Standard DNA Templates Standard DNA Templates STR Locus K562 9947A 9948 D16S539 12 11 12 11 11 11 D7S820 11 9 11 10 11 11 D13S317 8 8 11 11 11 11 D58818 12 11 11 11 13 11 CSF1PO 10 9 12 10 12 11 10 TPOX 9 8 8 8 9 8 Amelogenin X X X X X Y TH O 9393 2938 2936 vWA 16 16 18 17 17 17 1Strain 9948 displays three alleles at the CSF1PO locus Il C Power of Discrimination The eight STR loci amplified with the PowerPlex 1 2 System provide powerful discrimination Population statistics for these loci and their various multiplex combinations are dis
47. lic Ladders Product Size Cat Internal Lane Standard 600 150ul DG2611 GammaSTR Allelic Ladder Mix Fluorescein 150ul DG3291 CTTv Allelic Ladder Mix Fluorescein 150yl DG2121 FFFL Allelic Ladder Mix Fluorescein 150yl DG2131 For Laboratory Use Accessory Components Product Size Cat PowerPlex Matrix Standards 310 377 50ul each dye DG3640 PowerPlex Matrix Standards 3100 Custom 10ul each dye X3121 PowerTyper Macros 1 CD ROM DG3470 Bromophenol Blue Loading Solution aml DV4371 3 x 1ml Fluorescent Ladder CXR 60 400 Bases 65yl DG6221 Gel Tracking Dye 1ml DV4361 4 x 250ul Gold ST R 10X Buffer 1 2ml DM241 1 Mineral Oil 12ml DY1151 Nuclease Free Water 50ml P1193 2 x 25ml Not for Medical Diagnostic Use For Laboratory Use Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Telephone 608 274 4330 Fax 608 277 2516 www promega com Part TMD009 Printed in USA Revised 6 07 Page 33 Promega XII B Related Products continued Sample Preparation Systems Product Size Cat DNA IQ System 100 reactions DC6701 400 reactions DC6700 AluQuant Human DNA Quantitation System 400 determinations DC1011 80 determinations DC1010 For Laboratory Use Not for Medical Diagnostic Use Polyacrylamide Gel Electrophoresis Reagents Product Size Cat Ammonium Persulfate 25g V3131 TBE Buffer 10X 1L V4251 Urea 1kg V3171 ART
48. lleles are below 200RFU or Distributor Contact the labeled peak the software will not be able information available at could not be found to locate the allele peak WWW promega com The THO1 9 3 allele Run the allelic ladder sample E mail was included in the without the THO1 9 3 allele techserv promega ladder sample com Error message The base pair size of Compare the size of the smallest Could not complete the alleles within the allele in the allelic ladder with the the Run Macro allelic ladder does not base pair size and range listed in command because not fall within the defined the same alleles If necessary no dye lanes are category range increase the range to greater than selected 4bp and save the new parameters Confirm that the internal lane standard fragments are correctly sized Redefine the internal lane standard fragments and reanalyze the sample using GeneScan software Allelic ladder data is not Confirm that the PowerTyper compatible with the Macro file matches the allelic PowerTyper file used ladder being used CE spikes in the allelic Use a different injection of the ladder sample are allelic ladder identified as alleles by the macro Allelic ladder peaks are Use a shorter injection time de too high causing stutter crease the amount of allelic ladder peaks to be called as used or reanalyze the allelic ladder allele peaks sample using increased peak ampli tude thresholds in the GeneScan
49. low and red dye colors To import the red channel data select Import from Preferences under the Edit menu 4 Double click on Check ILS macro The macros are listed at the bottom left corner of the active window A plots window will be displayed to show the internal lane standard i e Fluorescent Ladder CXH 60 400 Bases in the red dye color Scroll down to view and edit all injections lanes If neces sary use the GeneScan software to redefine the internal lane standard fragments and reanalyze the samples Note If the yellow dye color contains peak heights greater than 2 000RFU for the TMR labeled loci i e CTATv loci bleedthrough generally less than 150RFU will be observed in the red dye color This does not affect interpre tation and allele designations of loci in the blue and yellow dye colors 5 Double click on the POWER macro The offsets will be calculated for all loci When completed a plots window will open to display the blue dye allelic ladders i e GammaSTR System allelic ladders D16S539 D7S820 D13S317 and D58818 Confirm the correct allele numbers were assigned to the allelic ladders In general the allelic ladders contain fragments of the same lengths as either several or all known alleles for the locus The allelic ladder sizes and repeat units are listed in Table 1 Analysis using GeneScan analysis soft ware and Genotyper software allows allele determination by comparing amplified samp
50. mer Pair Mix 2 5yl AmpliTaq Gold DNA polymerase 0 45ul 2 25u master mix volume 22 5ul Per Tube template DNA volume to be added 2 5ul total reaction volume 25 ul 1Assumes the AmpliTaq Gold DNA polymerase is at 5u ul If the enzyme concentration is different the volume of enzyme used must be adjusted accordingly Note If the volume of AmpliTaq Gold DNA polymerase added to the master mix is less than 0 5ul you might wish to dilute the enzyme with 1X Gold ST R Buffer first and add a larger volume The amount of Nuclease Free Water in the reaction should be adjusted accordingly so that the final volume of master mix per reaction is 22 5ul Do not store diluted AmpliTaq Gold DNA polymerase 6 Add 22 5ul of PCR master mix to each reaction tube and place at room temperature or on crushed ice if using Tag DNA polymerase 7 Pipet 2 5ul 1ng template DNA of each sample into the respective tube containing 22 5ul of PCR master mix Note If template DNA is stored in TE buffer the volume of the DNA sample added should not exceed 20 of the final reaction volume PCR amplification efficiency and quality can be greatly altered by changes in pH due to added Tris HCl available magnesium concentration due to chelation by EDTA or other PCR inhibitors which might be present at low concentrations depending on the source of template DNA and extraction procedure used 8 Forthe positive amplification control dilute the K562 DNA to 0 4
51. ng ul Pipet 2 5ul 1ng of diluted K562 DNA into a microcentrifuge tube containing 22 5ul of PCR master mix 9 Forthe negative amplification control pipet 2 5ul of Nuclease Free Water instead of template DNA into a microcentrifuge reaction tube containing 22 5yl of the PCR master mix 10 If using the GeneAmp PCR system 2400 9600 or 9700 thermal cycler and l as l i oil to flow down the side MicroAmp reaction tubes no addition of mineral oil to the reaction tubes is ofthe tube and formari required However if using the model 480 thermal cycler and GeneAmp overlay to limit sample reaction tubes add 1 drop of mineral oil to each tube before closing loss or cross contamina tion due to splattering Note Allow the mineral Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Telephone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD009 Revised 6 07 Page 9 Promega D Storage of amplified samples at 4 C or higher can produce degradation products Note Amplification and detection instrumentation can vary You might need to optimize protocols including cycle number and injection time or loading volume for each laboratory instrument Note PowerPlex 1 2 is optimized for 1ng of DNA template In house validation should be performed V B Amplification Thermal Cycling Place the tubes in a thermal cycler 2 Select an
52. ons VI C Sample Preparation The Fluorescent Ladder CXR 60 400 Bases is included in the PowerPlex 1 2 System as the internal lane standard ILS for three color detection and analysis of amplified samples 1 Prepare a loading cocktail by combining the Fluorescent Ladder CXR and Hi Di formamide as follows 1ul Fluorescent Ladder x injections 24pl Hi Di formamide x injections Vortex for 10 15 seconds Combine 25ul of the prepared loading cocktail and 1ul of amplified sample Mix by pipetting Note Instrument detection limits vary therefore injection time or the amount of product mixed with loading cocktail might need to be increased or decreased If the peak heights are too high i e greater than 2 000HFU the samples can be diluted in Gold ST R 1X Buffer before mixing with load ing cocktail This might result in uneven allele peak heights across loci For best results use less DNA template in the amplification reactions 4 Combine 25ul of the prepared loading cocktail and 1 of the PowerPlex 1 2 Allelic Ladder Mix vortex the Allelic Ladder Mix prior to pipetting Note To run the THO1 Allele 9 3 alone prepare a 1 4 dilution 1 part THO1 Allele 9 3 with 3 parts 1X STR Buffer and combine 1ul of this dilution with 25ul of loading cocktail To run the THO1 Allele 9 3 in combination with the PowerPlex 1 2 Allelic Ladder prepare a 1 3 mixture 1 part THO1 Allele 9 3 with 3 parts PowerPle
53. ons for DNA Amplification 1989 Erlich H A ed Stockton Press New York NY PCH Protocols A Guide to Methods and Applications 1990 Innis M A et al eds Academic Press San Diego CA Bassam B J Caetano Anolles G and Gresshoff P M 1991 Fast and sensitive silver staining of DNA in polyacrylamide gels Anal Biochem 196 80 3 Budowle B et al 1991 Analysis of the VNTR locus D1S80 by the PCR followed by high resolution PAGE Am J Hum Genet 48 137 44 Nakamura Y et al 1987 Variable number of tandem repeat VNTR markers for human gene mapping Science 235 1616 22 Budowle B and Monson K L 1989 In Proceedings of an International Symposium on the Forensic Aspects of DNA Analysis Government Printing Office Washington DC Levinson G and Gutman G A 1987 Slipped strand mispairing A major mechanism for DNA sequence evolution Mol Biol Evol 4 203 21 Schlotterer C and Tautz D 1992 Slippage synthesis of simple sequence DNA Nucl Acids Hes 20 211 5 Smith J R et al 1995 Approach to genotyping errors caused by nontemplated nucleotide addition by Tag DNA polymerase Genome Hes 5 312 7 Magnuson V L et al 1996 Substrate nucleotide determined non templated addition of adenine by Taq DNA polymerase Implications for PCR based genotyping Bio Techniques 21 700 9 Walsh P S Fildes N J and Reynolds R 1996 Sequence analysis and charac
54. played in Tables 4 6 These data were developed as part of a collaboration 20 with Genetic Design Inc Greensboro NC Generation of these data included analysis of over two hundred individuals from African American Caucasian American and Hispanic American populations For additional population data for STR loci see references 21 25 Table 4 shows the matching probability 26 for the PowerPlex9 1 2 System in various populations The matching probability of the system ranges from 1 in 114 000 000 for Caucasian Americans to 1 in 274 000 000 for African Americans The matching probability of the PowerPlex 1 2 System in combination with the GenePrint Fluorescent STR Multiplex F13A01 F13B FESFPS LPL FFFL multi plex is 1 in 303 000 000 000 for Caucasian Americans and 1 in 4 610 000 000 000 for African Americans Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Telephone 608 274 4330 Fax 608 277 2516 www promega com Part TMD009 Page 4 Printed in USA Revised 6 07 A measure of discrimination often used in paternity analyses is the paternity index PI a means for presenting the genetic odds in favor of paternity given the geno types for the mother child and alleged father 27 The typical Pls for the PowerPlex 1 2 System and the PowerPlex 1 2 System in combination with the FFFL multiplex are shown in Table 5 The PowerPlex 1 2 System alone provides typical pa
55. ple tray and closing the doors select Run to start the capillary electrophoresis system Monitor the electrophoresis by observing the raw data and status windows Each sample will take approximately 30 minutes for syringe pumping sample injection and sample electrophoresis 4 Analyze the data using the GeneScan Analysis Software Amplified sample peak heights of less than 2 000HFU are ideal Note Peak heights greater than 2 000RFU might generate artifact peaks due to instrument saturation i e overloading the sample If the sample peak heights are not within the linear detection of the instrument the ratio of stutter peaks to real allele peaks increases and allele designations become difficult to interpret The balance of the peak heights might also appear less uniform Vil Detection of Amplified Fragments Using the ABI PRISM 3100 Genetic Analyzer Materials to Be Supplied by the User e dry heating block water bath or thermal cycler e crushed ice e aerosol resistant pipette tips e 3100 capillary array 36cm e performance optimized polymer 4 POP 4 for the 3100 e 10X genetic analyzer buffer with EDTA e sample tubes and septa for the 3100 e Hi Di formamide Applied Biosystems Cat 4311320 e PowerPlex9 Matrix Standards 3100 Custom Cat X3121 Q The quality of the formamide is critical Use Hi Di formamide with a conductivity lt 100uS cm Freeze formamide in aliquots at 20 C Multiple freeze thaw
56. poration 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Telephone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD009 Revised 6 07 Page 11 Note To resolve the 9 3 and 10 alleles the use of performance optimized polymer 6 POP 6 might be necessary n Caution Formamide is an irritant and a teratogen avoid inhalation and contact with skin Read the warning label and take the necessary precautions when handling this substance Always wear double gloves and safety glasses when working with formamide VI Detection of Amplified Fragments Using the ABI PRISM 310 Genetic Analyzer Materials to Be Supplied by the User Solution compositions are provided in Section XII A dry heating block water bath or thermal cycler 310 capillaries 47cm x 50um Applied Biosystems performance optimized polymer 4 POP 4 M Applied Biosystems glass syringe 1ml 10X genetic analyzer buffer with EDTA Applied Biosystems sample tubes and septa Applied Biosystems aerosol resistant pipette tips Section XII B Hi Di formamide Applied Biosystems Cat 4311320 PowerPlex Matrix Standards 310 377 Cat 4 DG3640 crushed ice Q The quality of the formamide is critical Use deionized formamide with a conductivity lt 100uS cm Freeze formamide in aliquots at 20 C Multiple freeze thaw cycles or long term storage at 4 C can cause a breakdown of the formamide
57. r cccssssssccecseeeeceeeeeeeeeeeeeeeeeeeeeeeees 12 A Matrix otandardizallOllssssssacuecinsteiues sex uten E deuvetes aed iuee em bueno qo en ec sum bE GG Fautn SUE 12 E Instrument Preparation sicrete enr etui dato edite icd sunm EEEE 12 C Sample PrepAardaliOlsucueducepat e rms cid iomiku s Uirense Et uax ei ea aasecamcsauseacddsededesdsuzese 13 D Capillary Electrophoresis and Detection ccccsccccsseeeseeeeceeeeseneeseneesaaees 14 VII Detection of Amplified Fragments Using the ABI PRISM 3100 Genetic Analyzer cccscscccscceeeceeeeeeeeeeeeeeeeeeeeeeeees 14 A Spectral Calibration occas LPT 14 B Sample FreparatiO RU mM 15 C Instrument Preparation cccsecccseececeeeeceeeeceeeeceueeeseeeceeeesegeeseueesseeeeseeeees 15 D Sample Detection ERIT 16 Vill Detection of Amplified Fragments Using the ABI PRISM 377 DNA Sequencer ccccccccccssssssssesssseseeseecceeceeeeeeeeeeeeeess 17 A Polyacrylamide Gel Preparation ccccccssceccesseecseeeecceeeeeceeeeeeseueesseaeeeess 18 B Matrix StandardizallQl secmiceete en ttoioqntetu eros Muy veces sede odes Scoti tini oduicebu i rens ct 19 C Instrument Preparation reino nrbe caine cor kieua nS pontes nu x8 vo Feci n e np ns 19 D CI Ncquc 19 E Sample Preparation and Loading cccccssccceececeeeeceeeeeseeeeceeeesegeesneessees 20 F Gel Electrophoresis and
58. ran Loading Solution TEMED corrosive flammable urea irritant Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Telephone 608 274 4330 Fax 608 277 2516 www promega com Part TMD009 Printed in USA Page 6 Revised 6 07 Product Components and Storage Conditions Product Size Cat PowerPlex 1 2 System 100 reactions DC6101 Not For Medical Diagnostic Use Cat DC6101 contains sufficient reagents for 100 reactions of 25ul each Includes o 300ul Gold ST R 10X Buffer o 250ul PowerPlex 1 2 10X Primer Pair Mix o 25ul PowerPlex 1 2 Allelic Ladder Mix o 100ul GenePrint THO1 Allele 9 3 TMR e 125ul Fluorescent Ladder CXR 60 400 Bases 3ug K562 DNA High Molecular Weight 10ng ul o 1ml Blue Dextran Loading Solution o 1 Protocol Storage Conditions Store all components at 20 C The PowerPlex 1 2 10X ns Allelic x Primer Pair Mix PowerPlex 1 2 Allelic Ladder Mix THO1 Allele 9 3 and ipu ANA UAR in a separate sealed Fluorescent Ladder CXR must be stored in the dark The postamplification Dac e i This components Allelic Ladder Mix THO1 Allele 9 3 Fluorescent Ladder CXR and component should be Loading Solution are packaged separately to prevent cross contamination We moved to the post strongly recommend that pre amplification and postamplification reagents be stored amplification box after and used separately with different pipettes tube racks e
59. romega com Printed in USA Part TMD009 Revised 6 07 Page 27 X Troubleshooting continued X B PowerTyper 1 2 Macro Symptoms Possible Causes Comments File does not open Genotyper software Be certain that Genotyper 2 0 or not installed later version is installed on your computer Incorrect version of The PowerTyper 1 2 Macro Genotyper software might not work with versions prior to Genotyper version 2 0 Damaged CD ROM The CD ROM might have been damaged during shipment Contact Technical Services at 800 356 9526 or 608 274 4330 Error message Allelic ladder sample Be certain the sample info or Could not complete files have not been color info column for each lane the Run Macro identified containing the PowerPlex 1 2 command because Allelic Ladder Mix contains the no dye lanes are word ladder to identify the sam selected ple files containing allelic ladder All four dye colors not For Genotyper9 2 5 set the imported preferences under edit to import the blue green yellow and red colors The plots window or The macros were not The Check ILS macro and Power allele table does not run in the proper order macro must be run first before the display all the data remaining macros are run All four dye colors not For Genotyper 2 5 or higher set imported the preferences under edit to import the blue green yellow and red colors The Check ILS All four dye colors not For Genotyper 2
60. ross contamination can be a in one or all of the another template DNA problem Use aerosol resistant color channels or previously amplified pipette tips and change gloves DNA regularly Artifacts of STR PCR amplification of STR systems amplification sometimes generates artifacts that appear as faint peaks one or four bases below an allele Stutter band peak heights will be high if the samples are overloaded i e gt 2000RFU Samples not completely Heat denature the samples for the denatured recommended time and cool on ice immediately prior to loading the gel or capillary Background Load less amplification product or decrease the injection time Capillary electro Minor voltage changes or urea phoresis CE related crystals passing by the laser can artifacts spikes cause spikes or unexpected peaks Spikes sometimes appear in one color but often are easily identified by their presence in more than one color Re inject the samples to confirm CE related artifacts Contaminants in the water used contaminants on the ABI PRISM 310 and 3100 Genetic Analyzers and to dilute the 10X genetic analyzer buffer can generate peaks in the blue and green dye colors Use autoclaved water change vials and wash buffer reservoir Excessive amount of We recommend 1 2ng of DNA DNA template Amplification of DNA templates gt 2ng can result in a higher number of stutter bands Use less DNA template or reduce the number o
61. s The PowerTyper 1 2 Macro is used in conjunction with Genotyper 2 0 or 2 5 Macintosh and Genotyper 3 6 Windows software Therefore the appropri ate version of Genotyper software must be installed on your computer Amplified sample peak heights of less than 2 000RFU are ideal Be certain the sample info column for each lane containing the PowerPlex 1 2 Allelic Ladder Mix contains the word ladder The macro uses the word ladder to identify the sample files containing allelic ladder Sample info can be added or modified after importing into PowerTyper M Macro Highlight the sample then select show dye lanes window under views Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Telephone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD009 Revised 6 07 Page 21 Q Important The transferred file can be overwritten if save as is not used This can be avoided by locking the file The PowerPlex 1 2 System contains a vial of GenePrint THO1 Allele 9 3 TMR to enable the user to evaluate the resolution of THO1 alleles 9 3 and 10 using their conditions and detection instrumentation The PowerTyper M 1 2 Macro uses the PowerPlex 1 2 Allelic Ladder Mix without the THO1 Allele 9 3 for determining sample genotypes While the allelic ladder mix does not contain THO1 Allele 9 3 the macro will identify samples containing
62. tc opening Product Cat Powerlyper Macros DG3470 Not For Medical Diagnostic Use The PowerTyper Macros for use with Genotyper software are available from Promega This CD ROM contains the file PowerTyper 1 2 Macro for use with the PowerPlex 1 2 System The Macros can also be downloaded at www promega com geneticidtools DNA Extraction and Quantification Methods The DNA IQ System Cat DC6701 is a DNA isolation and quantitation system designed specifically for forensic and paternity samples 30 This novel system uses paramagnetic particles to prepare clean samples for STR analysis easily and effi ciently and can be used to extract DNA from stains or liquid samples such as blood or solutions The DNA IQ Resin is designed to eliminate PCR inhibitors and contami nants frequently encountered in casework samples For larger samples the DNA IQ System delivers a consistent amount of total DNA The system has been used to isolate and quantify DNA from routine sample types including buccal swabs stains on FTA paper and liquid blood Additionally DNA has been isolated from casework samples such as tissue differentially separated sexual assault samples and stains on support materials The DNA IQ System has been tested with the PowerPlex oystems to ensure a streamlined process See Section XII B for ordering information For applications requiring human specific DNA quantification the AluQuant Human DNA
63. ternity indices exceeding 260 in each group enough to satisfy routine requirements for paternity determination When the FFFL multiplex is also included the values exceed 2 600 in all groups An alternative calculation used in paternity analyses is the power of exclusion 27 This value calculated for the PowerPlex 1 2 System exceeds 0 9969 in all popula tions tested Table 6 In combination with the FFFL multiplex the values exceed 0 99974 demonstrating the usefulness of these two systems for paternity analyses as well as for forensic determinations Table 4 Matching Probabilities of the PowerPlex 1 2 System in Various Populations Matching Probability African Caucasian Hispanic STR System American American American PowerPlex 1 2 System 1 in 1 in 1 in 8 STR loci 2 74 x 108 1 14 x 108 1 45 x 108 PowerPlex 1 2 System 1 in 1 in 1 in combined with FFFL 4 61 x 1012 3 03 x 1011 4 75 x 1011 multiplex 12 STR loci Table 5 Typical Paternity Indices of the PowerPlex 1 2 System in Various Populations Typical Paternity Index African Caucasian Hispanic STR System American American American PowerPlex 1 2 System 498 260 319 PowerPlex 1 2 System 8 373 3 976 2 627 combined with FFFL multiplex Table 6 Power of Exclusion of the PowerPlex 1 2 System in Various Populations Power of Exclusion African Caucasian Hispanic STR System American American American PowerPlex 1 2 System 0 9982 0 9969 0 9973 8
64. the urea from the well area Load ipl of each denatured sample into the respective wells are recommended 10 Place the lid on the upper buffer chamber and close the instrument door Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Telephone 608 274 4330 Fax 608 277 2516 www promega com Part TMD009 Page 20 Printed in USA Revised 6 07 Promega VIII F Gel Electrophoresis and Detection 1 After loading select Cancel to stop the prerun Make sure that the run time is set at 2 5 hours then select Run to begin electrophoresis 2 Monitor the electrophoresis by observing the gel image and status windows Allow electrophoresis to proceed for 2 5 hours The 400 base fragment will have migrated past the laser 4 Analyze the gel according to the GeneScan Analysis Software User s Manual Amplified sample peak heights of less than 2 000HFU are ideal Notes e Peak heights greater than 2 000RFU can generate artifact peaks due to instrument saturation i e overloading the sample If the sample peak heights are not within the linear detection of the instrument the ratio of stutter peaks to real allele peaks increases and allele designations become difficult to interpret The balance of the peak heights might also appear less uniform e There might be variation between instruments regarding the relative fluores cence units detected using the same sample Furthermore the dif
65. those alleles We rec ommend mixing the THO1 Allele 9 3 with the PowerPlex 1 2 Allelic Ladder Mix Section VI C or VIII E and running this mixture in one lane to demonstrate that the separation of THO1 alleles 9 3 and 10 is detected using your parameters Follow the instructions below to determine allele designations for the nine loci D16S539 D7S820 D138317 D58818 CSF1PO TPOX Amelogenin THO1 and vWA contained in the PowerPlex 1 2 System Each macro must be com pleted in the proper order to correctly genotype the allelic ladders and samples Note Data generated on the ABI PRISM 3100 Genetic Analyzer is analyzed using a Windows NT version of the GeneScan analysis software This data can be imported directly into the Windows NT Genotyper version Before this data can be imported into the Macintosh version of Genotyper a conversion utility available from Applied Biosystems must be run This utility enables the Windows NT generated data to be recognized by the Macintosh software IX B Analysis 1 Transfer the PowerTyper 1 2 Macro from the PowerTyper M Macros CD ROM Cat DG3470 to a designated location on your computer hard drive 2 Open the Genotyper software and the PowerTyper 1 2 Macro For ques tions regarding the Genotyper software refer to the Genotyper User s Manual 3 Under File select Import to import the GeneScan project or sample files to be analyzed Import the blue yel
66. uggest strict adherence to recommended procedures for amplification as well as for denaturing gel electrophoresis and fluorescence detection STR analysis is subject to contamination by very small amounts of nontemplate human DNA Extreme care should be taken to avoid cross contamination when preparing sample DNA handling primer pairs setting up amplification reactions and analyzing amplification products Reagents and materials used prior to amplification Gold ST R 10X Buffer and PowerPlex 1 2 10X Primer Pair Mix should be stored separately from those used following amplification PowerPlex 1 2 Allelic Ladder Mix Fluorescent Ladder CXR and Loading Solution Always include a negative control reaction i e no template to detect reagent contamination We highly recommend the use of gloves and aerosol resistant pipette tips e g ART tips Section XII B Some of the reagents used in the analysis of STR products are potentially hazardous and should be handled accordingly Table 7 describes the potential hazards associated with such reagents Table 7 Hazardous Reagents Reagents for ABI PRISM 310 and 3100 Genetic Analyzers Hazard formamide irritant teratogen contained in the Blue Dextran Loading Solution Reagents for ABI PRISM 377 DNA Sequencer Hazard acrylamide suspected carcinogen Long Ranger gel solution toxic ammonium persulfate oxidizer corrosive formamide irritant teratogen contained in the Blue Dext
67. x 1 2 Allelic Ladder then combine 1ul of this mixture with 25ul of loading cocktail 5 Denature the samples and ladder by heating at 95 C for 3 minutes and immediately chill on crushed ice for 3 minutes Denature the samples just prior to loading Assemble the tubes in the appropriate autosampler tray 48 or 96 tube Place the autosampler tray in the instrument and close the instrument doors Q You might need to optimize injection time at Step 5 for individual instruments Injection times of 2 5 seconds are recommended for amplifi cations that contain 1ng of DNA template Note The PowerTyper 1 2 Macro is not compati ble with ladder samples containing the THO1 Allele 9 3 and will give an error message so that no alleles are called Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Telephone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Revised 6 07 Part TMD009 Page 13 Note To resolve the 9 3 and 10 alleles the use of performance optimized polymer 6 POP 6 might be necessary Q Caution Formamide is an irritant and a teratogen avoid inhalation and contact with skin Read the warning label and take the necessary precau tions when handling this substance Always wear double gloves and safety glasses when working with formamide VI D Capillary Electrophoresis and Detection 1 After loading the sam

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