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PowerPlex® 2.1 System

1. 16 To Troubleshooting occae rmsimam anm eee 20 OMEN Coco met eB 22 MEE REC TAIN PTT 25 A Ivana a DUDIHBD donc eoo dte Mou Tren IU UPPER ODE ONU 25 B Advantages of Using the Loci in the PowerPlex 2 1 System 25 MC DRIGEESCOBDO IR Produc costes rivum UD WR PUN canes Eie 28 D Power of DISCPUNDOLOEa nbi Ne ENCUENTRA 29 E Methods tor Polyach amide Gel REUSE cj aausesiernetrurP inte sanna er F DNA Extraction and Quantitation Methodis eene 99 The Internal Lane Standard UU suites esie tries tenentur aec totis 33 H Agarose Gel Electrophoresis of Amplification Products Optional 34 I Composition of Buffers and Solutions eser opor r eter ESI en 95 b Related Produch NNOREROR A 36 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD011 Revised 7 08 Page 1 Description SIR short tandem repeat loci consist of short repetitive sequence elements 3 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 may be detected using the polymerase chain reaction 5 8 Alleles of STR loci are differentiated by the number of copies of the repeat
2. 9 B Advantages of Using the Loci in the PowerPlex 2 1 System The loci included in the PowerPlex 2 1 System Tables 6 and 7 have been selected because they satisfy the needs of several major standardization bodies throughout the world INTERPOL the European police network has established a set of four STR loci FGA D21S11 TH01 and vWA as a pan European standard The European Network of Forensic Science Institutes ENFSI has completed a multilaboratory study of STR loci and recommend that European laboratories employ the following seven STR loci plus Amelogenin FGA THOI vWA D351358 D851179 D18551 and D21S11 The loci amplified in the PowerPlex 2 1 System include both of these STR standard sets The United States Federal Bureau of Investigation FBI has selected 13 STR loci to be typed prior to inclusion of sample profiles in or searching of the U S national database of convicted offender profiles CODIS COmbined DNA Index System Eight of the PowerPlex 2 1 System loci D18551 D21S11 THO1 D3S1358 FGA TPOX D8S1179 and vWA are included in this core set of 13 STR loci When used in combination with the PowerPlex 1 1 System all CODIS core loci can be analyzed in two amplification reactions Three of the same loci TH01 TPOX and vWA are amplified in both systems to minimize possibilities of undetected sample mix up when performing the two amplifications The PowerPlex 2 1 System also contains a low stutter highly
3. Fax 608 277 2516 www promega com Printed in USA Part TMD011 Revised 7 08 Page 23 e 8 References continued v 28 Promega 29 30 cle 02 33 34 35 36 o 38 39 40 41 42 Micka K et al 1996 Validation of multiplex polymorphic STR amplification sets developed for personal identification applications J Forensic Sci 41 582 90 Puers C et al 1993 Identification of repeat sequence heterogeneity at the polymorphic STR locus HUMTHO01 AATG n and reassignment of alleles in population analysis using a locus specific allelic ladder Am J Hum Genet 53 953 8 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 Bever R A and Creacy S 1995 Validation and utilization of commercially available STR multiplexes for parentage analysis In Proceedings from the Fifth International Symposium on Human Identification 1994 Promega Corporation 61 8 Sprecher C J et al 1996 General approach to analysis of polymorphic short tandem repeat loci BioTechniques 20 266 76 Lins A M et al 1996 Multiplex sets for the amplification of polymorphic short tandem repeat loci silver stain and fluorescent detection BioTechniques 20 882 9 Jones D A 1972 Blood samples Probability of discrimination J Forensic Sci Soc 12 355 9 Brenner C and Morris J W 1990 In Proceedings
4. V3131 V4251 V3171 Cat DY1051 DY1061 DY1071 DY1081 DY1101 DY1111 DY1121 DY1131 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Revised 7 08 Partt TMD011 Page 37 JSTR 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 covering the foundational PCR process expired on March 29 2005 U S Pat Nos 6 238 863 and 6 767 703 and Korean Pat No 691195 have been issued to Promega Corporation for materials and methods for identifying and analyzing intermediate tandem repeat DNA markers Other patents are pending U S Pat Nos 5 843 660 and 6 221 598 Australian Pat No 724531 Canadian Pat No 2 118 048 and Korean Pat No 290332 have been issued to Promega Corporation for multiplex amplification of STR loci Other patents are pending 4 The purchase of this product does not convey a license to use AmpliTaq Gold DNA polymerase You should purchase
5. www promega com Part TMD011 Printed in USA Page 36 Revised 7 08 Accessory Components Product Bromophenol Blue Loading Solution Gel Tracking Dye Gold ST R 10X Buffer Mineral Oil Nuclease Free Water For Laboratory Use Sample Preparation Systems Product DNA IQ System Differex System Maxwell 16 Instrument Size 3ml 3 x 1ml Iml 4 x 250gl 1 2ml 12ml 50ml 2 x 25ml DNA IQ Reference Sample Kit for Maxwell 16 DNA IQ Casework Sample Kit for Maxwell 16 Plexor HY System Slicprep 96 Device Not for Medical Diagnostic Use For Laboratory Use Size 100 reactions A00 reactions 50 samples 200 samples each 48 preps 48 preps 800 reactions 200 reactions 10 pack For Research Use Only Not for use in diagnostic procedures Polyacrylamide Gel Electrophoresis Reagents Product Ammonium Persulfate TBE Buffer 10X Urea ART Aerosol Resistant Tips Product ART 10 Ultramicro Pipet Tip ART 20E Ultramicro Pipet Tip ART 20P Pipet Tip ART GEL Gel Loading Pipet Tip ART 100 Pipet Tip ART 100E Pipet Tip ART 200 Pipet Tip ART 1000E Pipet Tip Volume 0 5 10p1 0 5 10p1 20ul 100p1 100p1 100p1 2001 1 000gl Size 25g 1L 1kg Size tips pack 960 960 960 960 960 960 960 800 Cat DV4371 DV4361 DM2411 DY1151 P1193 Cat DC6701 DC6700 DC6801 DC6800 AS2000 AS1040 AS1210 DC1000 DC1001 V1391 Cat
6. C Table 5 Instrument Parameters for the Hitachi FMBIO and FMBIO II Fluorescence A Scanners and PowerPlex 2 1 System Promega Hitachi FMBIO Hitachi FMBIO II Fluorescence Scanner Fluorescence Scanner Material Type acrylamide gel acrylamide gel Resolution Horizontal 150dpi 150dpi Vertical 150dpi 150dpi Rate 0 1024s line NA Repeat 1 time 256 times Gray Level Correction Type range range Cutoff Threshold Low Background 50 50 High Signal 1 1 100 505nm channel 80 585nm channel Reading Sensitivity 80 100 650nm channel Focusing Point NA 0 00mm NA not applicable Focusing point of 0 00mm is based on use of 5mm glass plates If using precast gels or thinner glass plates the focusing point may need to be adjusted 5 F Reuse of Glass Plates Separate the glass plates and discard the gel Clean glass plates with deionized water and 1 Liqui Nox detergent The use of Liqui Nox detergent is extremely important as other kinds of soap can build up on the glass plates This will result in low signal and high background on the gels If the glass plates have a build up of soap residue on them we recommend soaking in 10 sodium hydroxide for 1 hour and rinsing well in deionized water If bind silane is used to fix the gel to the smaller glass plate soak the plate in 10 sodium hydroxide for 1 hour or until the gel comes off the plate and clean as described Promega Corporation 2800 Woods Hollow Road Ma
7. 1 1 System but may be amplified using an additional system 9 D Power of Discrimination The nine STR loci amplified with the PowerPlex 2 1 System provide powerful discrimination Population statistics for these loci and their various multiplex combinations are displayed in Tables 10 12 These data were generated as part of a collaboration 26 with The Bode Technology Group Springfield VA Data generation included analysis of over 200 individuals from each of the three major racial and ethnic groups in the United States For additional population data for STR loci see references 27 and 29 33 Table 10 shows the matching probability 34 for the PowerPlex 2 1 System in various populations The matching probability of the PowerPlex 2 1 System ranges from 1 in 84 600 000 000 for Caucasian Americans to 1 in 300 000 000 000 for African Americans The matching probability of the PowerPlex 2 1 System in combination with the PowerPlex 1 1 System is 1 in 10 700 000 000 000 000 for Caucasian Americans and 1 in 41 900 000 000 000 000 for African Americans Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD011 Revised 7 08 Page 29 Promega 9 D Power of Discrimination continued A measure of discrimination often used in paternity analyses is the paternity index PI a means for presenting th
8. 80 100 120 140 160 180 200 225 250 275 300 325 350 375 400 425 450 475 500 550 and 600 bases in length Each fragment is labeled with carboxy X rhodamine CXR and may be detected separately as a third color in the presence of PowerPlex 2 1 amplified material using the Hitachi FMBIO and FMBIO II Fluorescence Imaging Systems The ILS 600 is designed for use in each gel lane to increase precision in analyses when using the PowerPlex 2 1 System This practice reduces the number of allelic ladder lanes needed per gel and therefore increases the number of lanes available for amplified sample materials A protocol for preparation and use of this internal lane standard is provided in section 5 C Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Partt TMD011 Revised 7 08 Page 33 C 9 H Agarose Gel Electrophoresis of Amplification Products Optional A This procedure is optional if PCR is performed routinely in your laboratory Promega Agarose gel electrophoresis can be used to rapidly confirm amplification success prior to performing polyacrylamide gel or capillary electrophoresis Materials to Be Supplied by the User Solution compositions are provided in Section 9 1 TAE 1X buffer agarose 5X loading solution ethidium bromide solution 0 5pg ml Prepare a 2 agarose gel approximate
9. AmpliTaq Gold DNA polymerase licensed for the forensic and human identity field directly from your authorized enzyme supplier 1999 2008 Promega Corporation All Rights Reserved GammaSTR GenePrint Maxwell Plexor and PowerPlex are registered trademarks of Promega Corporation Differex DNA IQ and Slicprep are trademarks of Promega Corporation AmpliTag 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 Ficoll is a registered trademark of GE Healthcare Bio sciences FMBIO is a registered trademark of Hitachi Software Engineering Company Ltd Freedom EVO is a registered trademark of Tecan AG Corporation FTA is a registered trademark of Flinders Technologies Pty Ltd and is licensed to Whatman GenBank is a registered trademark of the U S Dept of Health and Human Services Kimwipes is a registered trademark of Kimberly Clark 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 MicroAmp is a registered trademark of Applera Corporation Nalgene is a registered trademark of Nalge Nunc International Triton is a registered trademark of Union Carbide Chemicals and Plastics Technology Corporation Products may be covered by pending or issued patents or may ha
10. Based on size ranges for each locus Table 7 and migration characteristics of the dyes contained in the Gel Tracking Dye stop electrophoresis before the smallest locus i e D351358 has reached the bottom of the gel Note For an SA 43 gel run the gel until the leading edge of xylene cyanol is approximately 17 5cm from the bottom of the gel Run time will vary with the power supply used For a 5 Long Ranger gel run time is approximately 1 hour 30 minutes 5 E Detection 1 After electrophoresis remove the gel glass plate unit from the apparatus Remove the comb and side spacers but do not separate the glass plates 2 The plates must be thoroughly cleaned before scanning Clean both sides of the gel glass plate unit with deionized water and lint free paper Ethanol should not be used to clean the plate unit ethanol fluoresces and may be detected as background by the FMBIO instrument 3 Scan the gel according to the parameters listed in Table 5 Use the 505nm filter to detect fluorescein labeled fragments the 585nm filter to detect TMR labeled fragments and the 650nm filter to detect the Internal Lane Standard 600 Different laboratories may wish to modify these parameters according to their personal preferences Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD011 Revised 7 08 Page 13
11. Gels Component 5 Gel Final Concentration urea 18 0g 6M deionized water 26 0ml 10X TBE Buffer 5 0ml 1X 50 Long Ranger gel solution 5 0ml 5 total volume 50ml Note Long Ranger Singel Packs may also be used Note While standard 4 or 5 polyacrylamide gels can be used the performance of the Hitachi FMBIO II Fluorescence Imaging System band finding software is better when 5 Long Ranger acrylamide is used Filter the acrylamide solution through a 0 2 micron filter e g Nalgene tissue culture filter Degas the Long Ranger acrylamide for 5 minutes Pour the filtered acrylamide solution into a squeeze bottle Add the appropriate amounts of TEMED and 10 ammonium persulfate listed in Table 4 to the acrylamide solution and mix gently Table 4 Amounts of TEMED and 10 Ammonium Persulfate for 5 Long Ranger Polyacrylamide Gels Component 5 Long Ranger Gel 50ml TEMED 18 0g 10 ammonium persulfate 26 0ml Pour the gel by starting at the well end of the plates and carefully pouring the acrylamide between the horizontal glass plates Allow the solution to fill the top width of the plates Slightly tilt the plates to assist movement of the solution to the bottom of the plates while maintaining a constant flow of solution When the solution begins to flow out from the bottom position the plates horizontally Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9
12. Sequence analysis and characterization of stutter products at the tetranucleotide repeat locus vWA Nucleic Acids Res 24 2807 12 21 Moller A Meyer E and Brinkmann B 1994 Different types of structural variation in STRs HumFES FPS HumVWA and HumD21S11 Int J Leg Med 106 319 23 22 Brinkmann B Moller A and Wiegand P 1995 Structure of new mutations in 2 STR systems Int J Leg Med 107 201 3 23 Griffiths R et al 1998 New reference allelic ladders to improve allelic designation in a multiplex SIR system Int J Legal Med 111 267 72 24 B r W et al 1997 DNA recommendations Further report of the DNA Commission of the ISFH regarding the use of short tandem repeat systems Int J Legal Med 110 175 6 25 Gill P et al 1997 Considerations from the European DNA Profiling Group EDNAP concerning STR nomenclature Forensic Sci Int 87 185 92 26 Levadokou E N et al 2001 Allele frequencies for fourteen STR loci of the PowerPlex 1 1 and 2 1 multiplex systems and Penta D locus in Caucasians African Americans Hispanics and other populations of the United States of America and Brazil J Forensic Sci 46 736 61 27 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 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330
13. Tracking Dye 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 9 J Some of the reagents used in the analysis of STR products are potentially hazardous and should be handled accordingly Table 1 describes the potential hazards associated with such reagents Table 1 Hazardous Reagents Reagent Hazard acrylamide suspected carcinogen toxic ammonium persulfate oxidizer corrosive bisacrylamide toxic irritant formamide contained in the Bromophenol irritant teratogen Blue Loading Solution and Gel Tracking Dye TEMED corrosive flammable urea irritant Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Partt TMD011 Page 4 Printed in USA Revised 7 08 4 Protocols for DNA Amplification Using the PowerPlex 2 1 System y Materials to Be Supplied by the User model 480 or GeneAmp PCR System 9600 9700 or 2400 thermal cycler Promega Applied Biosystems e microcentrifuge 0 5ml GeneAmp or 0 2ml thin walled MicroAmp reaction tubes or MicroAmp optical 96 well reaction plate Applied Biosystems e 1 5ml amber colored microcentrifuge tubes Fisher Cat 05 402 26 e aerosol resistant pipette tips Section 9 J e AmpliTag Gold DNA polymerase Applied Biosys
14. five trimeric and tetrameric tandem repeat loci in four human population groups Genomics 12 241 53 4 Warne D et al 1991 Tetranucleotide repeat polymorphism at the human f actin related pseudogene 2 actbp2 detected using the polymerase chain reaction Nucleic Acids Res 19 6980 5 Ausubel F M et al 1996 Unit 15 The polymerase chain reaction In Current Protocols in Molecular Biology Vol 2 John Wiley and Sons NY Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD011 Page 22 Printed in USA Revised 7 08 6 Sambrook J Fritsch E F and Maniatis T 1989 Chapter 14 In vitro amplification of DNA by the lt polymerase chain reaction In Molecular Cloning A Laboratory Manual Second Edition Cold Spring e Harbor Laboratory Press Cold Spring Harbor New York 7 PCR Technology Principles and Applications for DNA Amplification 1989 Erlich H A ed Stockton Pro mega Press New York NY 8 PCR Protocols A Guide to Methods and Applications 1990 Innis M A et al eds Academic Press San Diego CA 9 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 10 Presley L A et al 1992 The implementation of the polymerase chain reaction PCR HLA DQ alpha typing by the FBI laborato
15. not planned for the same day the gel may be stored as described below after scanning Gels with samples may be stored up to 3 days with no problem Longer periods have not yet been attempted Different electrophoresis apparatus can produce slightly different running conditions so it is advisable to either use the same apparatus for both the forward and reverse electrophoresis to ensure that the previous samples are removed in the pre run or ensure that different apparatus give comparable results Gel Storage Following the final run of the day store gels by placing plastic wrap around the gel Paper towels wetted with water should be placed inside the plastic wrap at the top and bottom of the gel to help keep the gels from drying out Reuse of gels that are older than one week is not recommended Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD011 Page 32 Printed in USA Revised 7 08 9 F DNA Extraction and Quantitation Methods C The DNA IQ System Cat DC6700 is a DNA isolation and quantitation system designed specifically for forensic and paternity samples 36 This novel system uses paramagnetic particles to prepare clean samples for STR analysis easily and efficiently and can be used to extract DNA from stains or liquid samples such as blood or solutions The DNA IQ Resin eliminates PCR inhibitors
16. polymorphic pentanucleotide repeat locus Penta E This additional locus adds significantly to the discrimination power of the system making the PowerPlex 2 1 System a single amplification system with a power of exclusion sufficient to resolve nearly all paternity disputes definitively In addition the extremely low level of stutter seen with Penta E makes it an ideal locus to evaluate DNA mixtures often encountered in forensic casework Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD011 Revised 7 08 Page 25 Promega 9 B Advantages of Using the Loci in the PowerPlex 2 1 System continued We have carefully selected STR loci and primers to avoid or minimize artifacts including those associated with Tag DNA polymerase such as repeat slippage and terminal nucleotide addition as well as genetic artifacts called microvariant alleles Repeat slippage 16 17 sometimes called n 4 bands stutter or shadow bands is due to the loss of a repeat unit during DNA amplification somatic variation within the DNA or both The amount of this artifact observed depends primarily on the locus and DNA sequence being amplified Individual laboratories should develop independent guidelines regarding acceptable cutoff values for repeat slippage as well as standards for background level and allel
17. program must be run in 9600 ramp mode The ramp rates are set in the Ramp Rate Modification screen While viewing the cycling program navigate to the Ramp Rate Modification screen by selecting More then Modify On the Ramp Rate Modification screen the default rates for each step are 100 The rate under each hold step is the rate at which the temperature will change to that hold temperature Figure 1 shows the ramp rates for the GeneAmp PCR System 9700 thermal cycler The ramp mode is set after start has been selected for the thermal cycling run A Select Method Options screen appears Select 9600 ramp mode and enter the reaction volume 3 tmp 22 cycles 2976 7486MA Figure 1 The ramp rates for the GeneAmp PCR System 9700 thermal cycler Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMDO11 Page 8 Printed in USA Revised 7 08 5 Detection of Amplified Fragments Using the Hitachi FMBIO and FMBIO II Fluorescence Imaging Systems xe Materials to Be Supplied by the User Pro mega Solution compositions are provided in Section 9 1 e polyacrylamide gel electrophoresis apparatus power supply 4 000 volts e dry heating block water bath or thermal cyclers e squaretooth comb 35cm 60 wells cut in half for 30 wells gel 0 4mm thick Owl Scientific Cat 525 60A or vinyl doublefine sh
18. sequence contained within the amplified region and are distinguished from one another using radioactive silver stain or fluorescence detection following electrophoretic separation The PowerPlex 2 1 System allows co amplification and three color detection of nine STR loci The PowerPlex 2 1 System contains the loci Penta E D18551 D21S11 THO01 D391358 FGA TPOX D851179 and vWA One of the two primers for Penta E D18551 D21S11 TH01 and D351358 is labeled with fluorescein FL and one primer specific for FGA TPOX D8S1179 and vWA is labeled with carboxy tetramethylrhodamine TMR All nine loci are amplified simultaneously in a single tube and analyzed in a single gel lane The PowerPlex 16 Monoplex System Penta E Fluorescein Cat DC6591 is available to amplify the Penta E locus This monoplex system allows amplification of a single locus to confirm results obtained with the PowerPlex 16 System PowerPlex 16 BIO System or PowerPlex 2 1 System The monoplex systems also can be used to re amplify DNA samples when one or more of the loci do not amplify initially due to nonoptimal amplification conditions or poor DNA quality The PowerPlex 2 1 System is customized for use with the Hitachi FMBIO and FMBIO II Fluorescence Imaging Systems The PowerPlex 2 1 System provides all of the materials necessary for amplification of STR regions of purified genomic DNA except for AmpliTaq Gold DNA polymerase This manual contains sepa
19. sole purpose of the agarose gel is to confirm the PCR success Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD011 Page 34 Printed in USA Revised 7 08 9 I Composition of Buffers and Solutions 40 acylamide bis 19 1 Dissolve 380g of acrylamide and 20g of bisacrylamide in 500ml of deionized water Bring volume to 1 liter with deionized water 10 ammonium persulfate Add 0 5g of ammonium persulfate to 5ml of deionized water Use 200pl of 10 ammonium persulfate for each 30ml of acrylamide gel solution Store the remaining volume in 2001 aliquots at 20 C Bromophenol Blue Loading Solution 10mM NaOH 95 formamide 0 05 bromophenol blue 0 5M EDTA pH 8 0 stock 186 1g Na EDTA 2H O Add EDTA to 800ml of deionized water with vigorous stirring Adjust the pH to 8 0 with NaOH about 20 of NaOH pellets Dispense into aliquots and sterilize by autoclaving ethidium bromide solution 10mg ml 1 0g ethidium bromide Dissolve 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 Wear gloves when working with the dye and wear a mask when weighing it Gel Tracking Dye 10mM NaOH 95 formamide 0 05 bromophenol blue 0 05 xylene cyanol FF Gold S
20. the highly polymorphic Penta E locus does not display frequent microvariants Table 8 lists the PowerPlex 2 1 System alleles revealed in commonly available standard DNA templates Table 7 The PowerPlex 2 1 System Allelic Ladder Information Size Range of Allelic Repeat Numbers of Ladder Components Repeat Numbers of Allelic Alleles Not Present STR Locus Label bases Ladder Components in Allelic Ladder Penta E FL 379 474 5 24 20 3 D18551 FL 290 366 8 10 10 2 11 13 13 2 14 27 D21S11 FL 203 259 24 242 25 252 26 28 282 2929 2 00 00 2791 0 2 92 32 2 33 33 2 04 94 2 35 39 2 30 38 THO1 HE 156 195 4 9 9 3 10 11 13 3 D351358 FL 115 147 12 20 FGA TMR 322 444 16518 18 2 19 19 220202 PANDA GL UD PI e IER Ep PAY 242 29 20 2 26 00 32 43 2 44 2 45 2 46 2 TPOX TMR 262 290 6 13 D8S1179 TMR 203 247 7 18 vWA TMR 123 171 10 22 1The length of each allele in the allelic ladder has been confirmed by sequence analyses When using an internal lane standard such as the Internal Lane Standard 600 the calculated sizes of allelic ladder components may differ from those listed This occurs because different sequences in allelic ladder and ILS components may cause differences in migration The dye label also affects migration of alleles The D21511 alleles correspond to alleles 53 57 59 61 77 79 81 as defined by the Forensic Science Service using a different nomenclature The alleles listed are those with a fr
21. 18 STR loci in three amplification reactions Each STR locus contained in the GammaSTR CTTv and FFFL multiplexes is also available as a fluorescein labeled monoplex system Each of the fluorescent STR systems contains all the materials required to perform amplification reactions except for Tag DNA polymerase and sample DNA The corresponding allelic ladders are included with all systems The Fluorescent Ladder CXR 60 400 Bases is used as an internal lane standard ILS and is included with the PowerPlex 1 1 System The Internal Lane Standard 600 60 600 bases is included with the PowerPlex 2 1 System The internal lane standards may be purchased separately Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD011 Page 28 Printed in USA Revised 7 08 Table 9 Loci and Labels for the Fluorescent STR Muliplex Systems y CTTv FFFL GammaSTR PowerPlex 1 1 PowerPlex 2 1 STR Locus Multiplex Multiplex Multiplex System System Promega CFS1PO FL TMR TPOX FL TMR TMR THO1 FL TMR FL vWA FL TMR TMR F13A01 FL FESFPS FL F13B FL EPE FL D165539 FL FL D75820 FL FL D135317 FL FL D55818 El FL Amelogenin TMR Penta E FE D18551 FL DPATS TH FL D3S1358 FL FGA TMR D851179 TMR FL 5 terminal fluorescein label TMR 5 terminal carboxy tetramethylrhodamine label Amelogenin is not included in the PowerPlex
22. 526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD011 Page 10 Printed in USA Revised 7 08 8 Insert the squaretooth comb between the glass plates until the teeth are C almost completely inserted into the gel or insert one 14cm doublefine e 49 point sharkstooth comb straight side into the gel between the glass Pro plates 6mm of the comb should be between the two glass plates mega 9 Secure the comb with three evenly spaced clamps 10 Pour the remaining acrylamide solution into a disposable conical tube as a polymerization control Rinse the squeeze bottle including the spout with water 11 Allow polymerization to proceed for at least 1 hour Check the polymerization control to be sure that polymerization has occurred Note The gel may be stored overnight if a paper towel saturated with 1X TBE or water and plastic wrap are placed around the top and bottom of the gel to prevent the gel from drying out crystallization of the urea will destroy the gel 5 B Gel Pre Run 1 Remove clamps from the polymerized acrylamide gel and clean the glass plates with paper towels saturated with deionized water 2 Shave any excess polyacrylamide away from the comb and remove the comb 3 Add 1X TBE buffer to the bottom chamber of the electrophoresis apparatus 4 Gently lower the gel glass plates unit into the buffer with the longer plate facing out and the well side on top 5 Secure the glass plates to t
23. 6 base X specific band TMR carboxy tetramethylrhodamine FL fluorescein JOE 6 carboxy 47 5 dichloro 27 7 dimethoxy fluorescein NA not applicable Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD011 Page 26 Printed in USA Revised 7 08 Terminal nucleotide addition 18 19 occurs when Tag DNA polymerase adds a C nucleotide generally adenine to the 3 ends of amplified DNA fragments in a So template independent manner The efficiency with which this occurs varies with different primer sequences Thus an artifact band one base shorter than expected Promega i e missing the terminal addition is sometimes seen We have modified primer sequences and added a final extension step of 60 C for 30 minutes 20 to the amplification protocols to provide conditions for essentially complete terminal nucleotide addition when recommended amounts of template DNA are used The presence of microvariant alleles alleles differing from one another by lengths other than the repeat length complicates 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 21 22 For example the FGA and D21S11 loci are highly polymorphic and display numerous relatively common microvariants For reasons yet unknown
24. BIO II Fluorescence Imaging System A lane trace is shown to the right of each panel The lane traces show imbalance in the heterozygous alleles in several loci This occurs primarily because the two copies of each chromosome are not present in equal amounts within this cell line The K562 DNA also shows a three band pattern for the locus D21S11 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD011 Revised 7 08 Page 19 Promega 7 Troubleshooting For questions not addressed here please contact your local Promega Branch Office or Distributor Contact information available at www promega com E mail genetic promega com Symptoms Faint or absent allele bands Bands are fuzzy throughout the lanes n 1 bands are present Causes and Comments Impure template DNA Because of the small amount of template used this is rarely a problem Depending on the DNA extraction procedure used and the sample source inhibitors may be present in the DNA sample Diluting the template in TE buffer or water prior to amplification may improve results Insufficient template Use the recommended amount of template DNA Insufficient enzyme activity Use the recommended amount of AmpliTaq Gold DNA polymerase Check the expiration date on the tube label Incorrect amplification program Confirm the amplification
25. Results I Figures 2 5 show typical results achieved using the PowerPlex 2 1 System in Promega the three color detection format as described in Section 5 E A 505nm Scan Fluorescein B 585nm Scan TMR L123456L789101112L Mt 23 45 6478 91011128 i h 24 z SS SS 46 2 E Z 43 2 PentaE E amp M 314 2 a 5 x H FGA 17 D18551 TEZGM B 19 z i TPOX i5 d EM ud 6 po 18 D21911 Boe eB D8S1179 E 24 499 edi z 22 THO1 e wir 4 one a VWA D3S1358 E o Figure 2 The PowerPlex 2 1 System Twelve DNA samples lanes 1 12 were amplified with the PowerPlex 2 1 Primer Pair Mix using 1ng of DNA template Lanes labeled L contain allelic ladders for each of the nine loci contained in the PowerPlex 2 1 System Panel A A scan using a 505nm filter showing the fluorescein labeled loci Penta E D18551 D21S11 TH01 and D351358 Panel B A scan using a 585nm filter showing the TMR labeled loci FGA TPOX D8S1179 and vWA In panels A and B each allelic ladder is labeled to the right with the number of copies of the repeated sequence contained within its corresponding largest and smallest alleles Two sets 17 to 31 2 and 43 2 to 46 2 are shown for the locus FGA The rare allele 13 3 is also shown for the locus TH01 All materials were separated using a 5 denaturing Long Ranger polyacrylamide gel and detected using the Hi
26. TXR 10X Buffer 500mM KCI re mega 100mM Tris HCI pH 8 3 at 25 C 15mM MgCl 1 Triton X 100 2mM each dNTP 1 6mg ml BSA 5X loading solution 5 Ficoll 400 0 1 bromophenol blue 0 1 xylene cyanol 100mM EDTA Na EDTA 2H 0 10mM Tris HCI pH 7 5 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 the volume to 1 liter with deionized water TBE 10X buffer 107 8g Tris base 7 44g EDTA Na EDTA 2H O 55 0g boric acid Dissolve Tris base and EDTA in 800ml of deionized water Slowly add the boric acid and monitor the pH until the desired pH of 8 3 is obtained Bring the final volume to 1 liter with deionized water TE buffer 10mM Tris HCl 0 1mM EDTA pH 8 0 221g Tris base 0 037g EDTA Na EDTA 2H 0 Dissolve Tris base and EDTA in 900ml of deionized water Adjust to pH 8 0 with HCI Bring the final 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 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Revised 7 08 Partt TMD011 Page 35 C 9 J Related Products A Fluorescent STR Multiplex Systems Product Size Cat PowerPlex 16 Monoplex System Penta E Fluorescein 100 reactions DC6591 PowerPlex 16 Monoplex System Penta D JOE 100 re
27. Technical Manual PowerPlex 2 1 System INSTRUCTIONS FOR USE OF PRODUCTS DC6470 AND DC6471 PRINTED IN USA Revised 7 08 Part TMD011 PowerPlex 2 1 System O All technical literature is available on the Internet at www promega com tbs Please visit the web site to verify that you are using the most current version of this Technical Manual Please contact Promega Technical Services if you have questions on use of this system E mail genetic promega com L Desi p oiee E eee ene ee ee ee ee 2 2 Product Components and Storage Conditions sss 3 Bo Petore You yg ecco este i 4 Protocols for DNA Amplification Using the PowerPlex 2 1 System 5 Ae a 8121 8 aro S DO Nt E mene een Eo reerrenrer 5 ME Sonet n COM Bra cs Cycli cases cea T 7 5 Detection of Amplified Fragments Using the Hitachi FMBIO and FMBIO II Fluorescence Imaging Systems 9 A Polyacrylamide Gel Preparo Doe imr bas Pi reenter enter tee Pts teeter ttre 9 Be Gelre RON bosco RRR R 11 Ce campile reparation and LoaditG seisis 12 D GELE lec Kopio o ccce EHI terre tet yer UNUM EE 13 E Som M oe 15 LH Paor eie TU canescens een ees 14 6 Data Anal ySio s os deseo N RO EBOINIUUNUNDI MINNS PMM MM 15 A XonBelb E E E AR DD DEB cua eens 15 B Allelic Ladders eese eene teetn teneret tete tonta tn toten 15 EE o c c
28. USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Partt TMD011 Revised 7 08 Page 31 Promega 9 E Methods for Polyacrylamide Gel Reuse continued Plate Preparation 1 To ensure that the wells and gel remain firmly fixed to the plate treat both plates with methacryloxypropyltrimethoxysilane 9p in 3ml 0 5 acetic acid in 9575 ethanol 2 Remove excess solution with 4 washes of ethanol Only the top portion of the short plate needs to be treated if three or fewer runs were performed and the acrylamide solution was degassed Acrylamide Gel Solution Preparation See Section 5 A D for acrylamide gel preparation gel pre run sample preparation and gel electrophoresis Scanning Scan the gel on a Hitachi FMBIO II Fluorescence Imaging System leaving the plates spacers and comb intact Removal of First Run Samples from Gel and Second Pre Run Remove samples from the first run from the gel by reverse electrophoresis electrodes reversed Use new buffer for this reverse electrophoresis and increase the reverse run time to 15 30 minutes longer than the first run at the same wattage This electrophoresis serves to preheat the gel for the second run It is not necessary to change the running buffer again between reverse electrophoresis of the first run and loading of the second run simply rinse wells with running buffer and proceed with sample loading If another run is
29. actions DC6651 PowerPlex 1 1 System 100 reactions DC6091 400 reactions DC6090 GenePrint GammaSTR Multiplex Fluorescein 100 reactions DC6071 400 reactions DC6070 GenePrint Fluorescent STR Multiplex CSF1PO TPOX TH01 vWA Fluorescein CTTv Multiplex 100 reactions DC6301 400 reactions DC6300 GenePrint Fluorescent STR Multiplex F13A01 FESFPS F13B LPL Fluorescein FFFL Multiplex 100 reactions DC6311 400 reactions DC6310 PowerPlex 16 System 100 reactions DC 6581 400 reactions DC6530 PowerPlex ES System 100 reactions DC6731 400 reactions DC6730 PowerPlex Y System 50 reactions DC6761 200 reactions DC6760 Not for Medical Diagnostic Use GenePrint Sex Identification Systems Product Size Cat GenePrint Fluorescent Sex Identification System Amelogenin Fluorescein 100 reactions DC5171 GenePrint Fluorescent Sex Identification System Amelogenin TMR 100reactions DC6171 Not for Medical Diagnostic Use Allelic Ladders Product Size Cat Internal Lane Standard 600 150pl DG1701 Fluorescent Ladder CXR 60 400 Bases 6l DG6221 GammaSTR Allelic Ladder Mix Fluorescein 150pl DG3291 CTTv Allelic Ladder Mix Fluorescein 150pl DG2121 FFFL Allelic Ladder Mix Fluorescein 150pl DG2131 For Laboratory Use Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516
30. ances Impure template DNA Inhibitors that may be present in forensic samples can lead to allele dropout or imbalance Gel was not run long enough Run gel as long as possible without running the 100 base ILS fragment off the bottom of the gel If necessary run the gel for additional time after the first scan then scan the gel a second time to achieve better separation of larger alleles Scanning resolution was too low Default scanning resolution is 150dpi If necessary this resolution can be increased to 300dpi which should help sharpen bands Part of the spacers were scanned Rescan the gel being careful not to scan any portion of the spacers Focusing point may need to be adjusted Confirm that 5mm plates are being used Adjust focusing point if necessary Plates were improperly washed Improper washing of plates can cause a soap residue to build up on the plates which can cause background fluorescence Plate may be soaked in 1N NaOH to clean off residue Do not use ethanol to clean plates prior to scanning 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 2 Edwards A et al 1991 DNA typing and genetic mapping with trimeric and tetrameric tandem repeats Am J Hum Genet 49 746 56 3 Edwards A et al 1992 Genetic variation at
31. and contaminants frequently encountered in casework samples With 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 Systems to ensure a streamlined process See Section 9 J for ordering information Promega For applications requiring human specific DNA quantification the Plexor HY System Cat DC1000 has been developed 37 See Section 9 J for ordering information The DNA IQ System has been fully automated on the Beckman Coulter Biomek 2000 Laboratory Automation Workstation 38 Biomek 3000 Laboratory Automation Workstation 39 and Tecan Freedom EVO Liquid Handler 40 In addition the DNA IQ Reference Sample Kit for Maxwell 16 Cat AS1040 and DNA IQ Casework Sample Kit for Maxwell 16 are available 41 42 For information about automation of laboratory processes on automated workstations contact your local Promega Branch Office or Distributor contact information available at www promega com worldwide or e mail geneticOpromega com 9 G The Internal Lane Standard 600 The Internal Lane Standard ILS 600 contains 22 DNA fragments of 60
32. arkstooth comb 14cm 49 point 0 4mm thick e Nalgene tissue culture filter 0 2 micron e aerosol resistant pipette tips Section 9 J e low fluorescence glass plates 43cm x 19cm x 0 4mm The Gel Company Cat GG047 B05055 e spacers 0 4mm e 5A 43 extension Lab Repco Cat 31096423 for use with 43cm glass plates e clamps e g large office binder clamps e diamond pencil for marking glass plates e 50 Long Ranger gel solution or Long Ranger Singel pack for ABI sequencers 377 36cm Cambrex e TBE 10X buffer 10 Ammonium Persulfate Cat V3131 e Urea Cat V3171 e TEMED e bind silane methacryloxypropyltrimethoxysilane for use with squaretooth combs e Ligui Nox detergent 5 A Polyacrylamide Gel Preparation O Caution Acrylamide is a neurotoxin and suspected carcinogen avoid inhalation and contact with skin Read the warning label and take appropriate precautions when handling this substance Always wear gloves and safety glasses when working with acrylamide solutions Note To reduce the time of preparing gels or the expense of precast gels we have developed a rapid method to reuse gels between 2 and 8 times over a period of several days 12 Methods for polyacrylamide gel reuse are provided in Section 9 E 1 Etch each glass plate on one side in one corner with a diamond pencil to distinguish the gel sides of the glass plates Thoroughly clean the glass plates twice with 95 ethanol and Kimwipes ti
33. at color separation Rerun gel with less sample DNA Gel not run in reverse long enough when reusing gels Gels should be run in reverse 15 30 minutes longer than the previous run Longer time may be required if using a different gel rig or power supply Allelic ladder contamination Keep pre and postamplification components separate Imbalance of band Excessive amount of DNA The system is balanced using 1ng intensities across loci of DNA template Amplification of 5ng of template can result in an imbalance with smaller loci showing more product than larger loci Use less template or reduce the number of cycles in the amplification program Too much template from cards or membrane punches of bloodstains Cards or membranes that bind DNA tightly can contain more template DNA than recommended This will lead to overrepresented smaller alleles and underrepresented larger alleles Use the recommended amount of template by using a smaller punch of the membrane Alternately fewer cyles of amplification can compensate for this type of unevenness of product yield i e 10 18 or 10 16 cycling The band intensity of the Penta E locus is generally about 6576 the intensity of the other loci Use of twice the recommended amount of AmpliTaq Gold DNA polymerase can compensate for the lower yield of the Penta E locus Poor quality polyacrylamide gel Prepare acryamide and buffer solutions using high quality reagents We recommend 576 Long Ranger acrylami
34. de Cambrex Too many cycles in the amplification protocol Use the recommended amplification program confirm the number of cycles Too much enzyme was present Use the recommended amount of AmpliTaq Gold DNA polymerase Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD011 Revised 7 08 Page 21 Promega 7 Troubleshooting continued Symptoms Imbalance of band intensities across loci continued Poor separation of alleles in ladder lanes or difficulty resolving microvariant alleles White background with low signal intensity Dark grainy background with low signal intensity 8 References Causes and Comments Degraded DNA sample DNA template is degraded and the larger loci show diminished yield Confirm the DNA integrity by running an aliquot on an agarose gel Repurify the template DNA if necessary Insufficient template DNA Use the recommended amount of template DNA Stochastic effects can occur when amplifying low amounts of template Miscellaneous balance problems Thaw the 10X Primer Pair Mix and Gold ST R 10X Buffer completely and vortex for 15 seconds before using Do not centrifuge the 10X Primer Pair Mix after mixing Calibrate thermal cyclers and pipettes routinely Using a 59 C annealing temperature instead of 60 C has been shown to improve balance in some inst
35. dison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD011 Printed in USA Page 14 Revised 7 08 6 Data Analysis C s View and analyze the gel image to determine allele designations as recommended in the FMBIO user s manual For information regarding the use of FMBIO Analysis Pro mega Software contact the Hitachi Technical Support Group 800 624 6176 extension 7508 Perform the multicolor separation for all gels containing material amplified using the PowerPlex 2 1 System Display the gel image using green for the fluorescein labeled loci Penta E D18551 D21511 TH01 and D3S1358 red for the TMR labeled loci FGA TPOX D8S1179 and vW A and cyan for the Internal Lane Standard 600 6 A Controls Observe the lanes containing the negative controls They should be devoid of amplification products Observe the lanes containing the K562 DNA positive controls Compare K562 DNA allelic repeat sizes with the locus specific allelic ladder The expected K562 DNA allele sizes for each locus are listed in Table 8 Section 9 B Figure 5 shows an example of results obtained after amplification of K562 DNA using the PowerPlex 2 1 System K562 DNA contains imbalanced alleles at several loci due to the unusual chromosome content of this cell line This is not a function of PowerPlex 2 1 System performance 6 B Allelic Ladders In general allelic ladders contain fra
36. e intensity The average percent of repeat slippage stutter for each locus present in the PowerPlex 2 1 System has been determined Table 6 Note the comparatively low stutter of Penta E Table 6 The PowerPlex 2 1 System Locus Specific Information STR Chromosomal GenBank Locus and Repeat Sequence Average Locus Label Location Locus Definition 5 3 Percent Stutter PentaE FL 15q NA AAAGA 1 275 D18551 FL 18q21 3 HUMUT574 AGAA 23 265 D21S11 FL 21q11 21q21 HUMD21LOC TCTA Complex 23 gt 6 THO1 PL 11p15 5 HUMTHOI human tyrosine AATG 23 1 275 hydroxylase gene D351358 FL 3p NA TCTA Complex gt 6 FGA TMR 4q28 HUMFIBRA human TUC 4 6 fibrinogen alpha chain gene Complex 23 TPOX TMR 2p24 2pter HUMTPOX human thyroid AATG 2 4 peroxidase gene D851179 TMR 8q NA TCTA Complex 23 4 6 vWA TMR 12p12 pter HUMVWEFA31 human von TCTA gt 6 Willebrand factor gene Complex 23 1The August 1997 report 24 25 of the DNA Commission of the International Society for Forensic Haemogenetics ISFH states 1 for STR loci within coding genes the coding strand shall be used and the repeat sequence motif defined using the first possible 5 nucleotide of a repeat motif and 2 for STR loci not associated with a coding gene the first database entry or original literature description shall be used Amelogenin is not an STR but displays a 106 base X specific band and a 112 base Y specific band 9947A DNA female displays only the 10
37. e genetic odds in favor of paternity given the genotypes for the mother child and alleged father 35 The typical paternity indices for the PowerPlex 2 1 System and PowerPlex 1 1 System in combination with the FFFL multiplex are shown in Table 11 The PowerPlex 2 1 System alone provides typical paternity indices exceeding 3 250 in each population group enough to satisfy routine requirements for paternity determination When the PowerPlex 1 1 System and PowerPlex 2 1 System are combined the values exceed 163 000 in all groups Table 10 Matching Probabilities of the PowerPlex Systems in Various Populations STR System PowerPlex 1 1 System 8 STR loci PowerPlex 2 1 System 9 STR loci PowerPlex 1 1 System plus PowerPlex 2 1 System 14 STR loci PowerPlex 1 1 System System plus PowerPlex 2 1 System plus FFFL muliplex 18 SIR loci African American in 2 74 x 108 Tru od es TE 1 in 4 19 x 10 6 LET A x TUA Matching Probability Caucasian Hispanic American American in 1 14 x 108 1 in 8 46 x 10 1 in 1 07 x 10 6 1 in 2 84 x 10 in 1 45 x 108 1in 1 02 x 10H in 1 59 x 10 6 Tit SpA o8 T Asian American in 1 32 x 105 Tin Wey xou in 2 17 x10 1 in 1 14 x 10 5 Table 11 Typical Paternity Indices of the PowerPlex Systems in Various Populations STR System PowerPlex 1 1 System 8 STR loci PowerPlex 2 1 System 9 STR loci PowerPlex 1 1 Syst
38. ed and the PowerPlex 2 1 Allelic Ladder Mix is loaded as often as every third gel lane 1 Prepare amplified samples and ladders as described below depending on the detection method employed either three color detection or two color detection For three color detection using the Internal Lane Standard 600 a Prepare a loading cocktail by combining and mixing Internal Lane Standard 600 and Bromophenol Blue Loading Solution as follows 1pl ILS 600 x amp lanes 3pl Bromophenol Blue Loading Solution x t lanes Combine 4 of prepared loading cocktail and 2yl of amplified sample Note If the fluorescent signal on the gel is too intense dilute samples in Gold STXR 1X Buffer before mixing with loading cocktail or use less DNA template in the amplification reactions Combine 4pl of prepared loading cocktail and 2yl of PowerPlex 2 1 Allelic Ladder Mix For two color detection not using the Internal Lane Standard 600 d Combine 2 5yl of Bromophenol Blue Loading Solution and 2 5pl of amplified sample Note If the fluorescent signal is too intense dilute samples in Gold ST R 1X Buffer before mixing with loading solution or use less DNA template in the amplification reactions Combine 2 5yl of Bromophenol Blue Loading Solution and 2 5pl of PowerPlex 2 1 Allelic Ladder Mix Optional Place 6pl of Gel Tracking Dye in one tube The Gel Tracking Dye contains both bromophenol blue and xylene cyanol This d
39. em plus PowerPlex 2 1 System 14 STR loci PowerPlex 1 1 System System plus PowerPlex 2 1 System plus FFFL African American 498 13 130 6 11 x 10 1 03 x 107 Typical Paternity Index Caucasian Hispanic American American 260 319 13 199 3 250 4 08 x 105 1 63 x 105 6 24 x 106 1 34 x 106 Asian American 471 41 800 2 02 x 106 4 55 x 106 Multiplex 18 STR loci Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD011 Page 30 Printed in USA Revised 7 08 An alternative calculation used in paternity analyses is the power of exclusion 35 This value calculated for the PowerPlex 2 1 System exceeds 0 9997 in all ke populations tested Table 12 In combination with the PowerPlex 1 1 System Pre the values exceed 0 9999951 demonstrating the usefulness of these two systems un g for paternity analyses as well as for forensic determinations Note The data in Tables 10 11 and 12 for the PowerPlex 1 1 System and FFFL multiplex were published in Lins et al 26 Data for the PowerPlex 2 1 System were generated as part of a collaborative effort between The Bode Technology Group and Promega Corporation Table 12 Power of Exclusion of the PowerPlex Systems in Various Populations Power of Exclusion African Caucasian Hispanic STR System American American America
40. equency of gt 1 1000 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Partt TMD011 Revised 7 08 Page 27 Table 8 The PowerPlex 2 1 System Allele Determinations in Commonly Available Standard DNA Templates Standard DNA Templates STR Locus K562 9947A 9948 Penta E 5 14 12 13 11 11 D18551 ile AE I5 319 15 18 D21511 293031 30 30 29 30 TH01 93 93 8 9 3 009 D351358 16 16 14 15 I5 FGA 21 24 23 24 24 26 TPOX 8 9 8 8 8 9 D851179 105 102 13 09 12 15 vWA 16 16 17 18 I d Strains 9947A and 9948 are available from the NIGMS Human Genetic Mutant Cell Repository Cornell Institute Camden NJ Strain K562 is available from the American Type Culture Collection Manassas VA 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 9 C Fluorescent STR Products Table 9 lists the fluorescent STR multiplex systems available from Promega Three quadriplexes the GammaSTR CTTv and FFFL multiplexes have been developed with fluorescein labels The combination of the PowerPlex 1 1 System 26 27 and PowerPlex 2 1 System provides analysis of 14 STR loci Use of the PowerPlex 1 1 System the PowerPlex 2 1 System and the FFFL System 26 27 provides analysis of
41. from the International Symposium on Human Identification 1989 Promega Corporation 21 53 Mandrekar P V Krenke B E and Tereba A 2001 DNA IQ The intelligent way to purify DNA Profiles in DNA 4 3 16 Krenke B E et al 2005 Development of a novel fluorescent two primer approach to quantitative PCR Profiles in DNA 8 1 3 5 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 McLaren B Bjerke M and Tereba A 2006 Automating the DNA IQ System on the Biomek 3000 Laboratory Automation Workstation Profiles in DNA 9 1 11 13 Cowan C 2006 The DNA IQ System on the Tecan Freedom EVO 100 Profiles in DNA 9 1 8 10 Bjerke M et al 2006 Forensic application of the Maxwell 16 Instrument Profiles in DNA 9 1 3 5 Mandrekar P et al 2007 Introduction to Maxwell 16 low elution volume configuration for forensic casework Profiles in DNA 10 2 10 12 The fields of forensic and paternity analysis are changing rapidly For this reason it is difficult to keep our manuals up to date regarding new technologies and new publications However a substantial reference list of publications describing STRs and much related information can be found at an Internet web site created by the National Institutes of Science and Technology NIST Biotechnology Division www cstl nist gov biotech strbase This web si
42. gments of the same lengths as most or all known alleles for the locus Allelic ladder sizes and repeat units are listed in Table 7 Section 9 B Visual comparison between allelic ladder and amplified samples of the same locus allows precise assignment of alleles Analysis using specific instrumentation also allows allele determination by comparing amplified sample fragments with either allelic ladders internal size standards or both see software documentation from instrument manufacturer When using an internal lane standard the calculated lengths of allelic ladder components will differ from those listed in Table 7 This is due to differences in migration resulting from sequence differences between allelic ladder fragments and internal lane standard fragments Note It may prove helpful to confirm that your gel analysis software identifies the correct number of alleles present in the allelic ladder lanes prior to analysis of sample lanes The PowerPlex 2 1 System Allelic Ladder has 86 alleles in the fluorescein channel 20 Penta E alleles 22 D18551 alleles 25 D21511 alleles 10 TH01 alleles and 9 D351358 alleles and 52 alleles in the TMR channel 19 FGA alleles 8 TPOX alleles 12 D8S1179 alleles and 13 vWA alleles Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Partt TMD011 Revised 7 08 Page 15 C 6 C
43. he gel electrophoresis apparatus 6 Add 1X TBE buffer to the top buffer chamber of the electrophoresis apparatus 7 Using a 50 100cc syringe filled with buffer remove the air bubbles on the top of the gel Be certain the well area is devoid of air bubbles and small pieces of polyacrylamide Using a syringe with a bent 18 gauge needle remove any air bubbles from the bottom of the gel 8 Pre run the gel to achieve a gel surface temperature of approximately 50 C Consult the manufacturer s instruction manual for the recommended electrophoresis conditions Note As a reference we generally use 60 watts for 45 60 minutes for a 43cm gel The gel running conditions may need to be adjusted to reach a temperature of 50 C 9 Prepare samples and allelic ladder samples during the gel pre run Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Partt TMD011 Revised 7 08 Page 11 Promega 5 C Sample Preparation and Loading The Internal Lane Standard 600 is included in the PowerPlex 2 1 System for those who wish to use an internal size marker for three color detection and analysis of amplified samples With this approach only 2 3 lanes of the PowerPlex 2 1 Allelic Ladder are required per gel Alternatively the two color detection method may be employed in which the Internal Lane Standard 600 is not us
44. hen 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 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 3 tmp 10 cycles Protocol for the 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 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 Model 480 Thermal Cycler 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 When using the GeneAmp PCR System 9700 thermal cycler the ramp rates indicated in the cycling program must be set and the
45. ions continued Available Separately Product Size Cat Internal Lane Standard 600 150yl DG1701 For Laboratory Use Additional fluorescent STR multiplex product information and ordering information for accessory components and related products is provided in Section 9 J and is available on the Internet at www promega com or upon request from Promega Before You Begin 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 10 11 The quality of the purified DNA as well as small changes in buffers ionic strength primer concentrations choice of thermal cycler and thermal cycling conditions can affect PCR success We suggest strict adherence to recommended procedures for amplification as well as electrophoresis and fluorescence detection PCR based 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 assembling amplification reactions and analyzing amplification products Reagents and materials used prior to amplification Gold ST R 10X Buffer PowerPlex 2 1 10X Primer Pair Mix and K562 DNA High Molecular Weight should be stored separately from those used following amplification PowerPlex 2 1 Allelic Ladder Mix Internal Lane Standard 600 Bromophenol Blue Loading Solution and Gel
46. late DNA used or reduce the number of cycles by 2 or 4 cycles 10 18 or 10 16 cycles Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD011 Page 20 Printed in USA Revised 7 08 Symptoms Causes and Comments C Extra bands visible in Contamination with another template DNA or previously e one or all lanes amplified DNA Cross contamination can be a problem Use aerosol resistant pipette tips and change gloves regularly Promega Samples were not completely denatured Heat denature samples for the recommended time and cool on crushed ice or in an ice water bath immediately prior to loading the gel Artifacts of STR amplification PCR amplification of STR systems sometimes generates artifacts that appear as faint bands one repeat unit smaller than the allele Refer to Section 9 B for locus specific information regarding this event Stutter band intensities can be high if samples are overloaded Use 1 2ng DNA template Artifacts of STR amplification PCR amplification of STR systems can result in artifacts that appear as bands one base smaller than the allele due to incomplete addition of the 3 A residue Be sure to perform the 30 minute extension step at 60 C after thermal cycling Section 4 B High background Load less amplification product Bleedthrough due to poor color separation If samples lanes are dark repe
47. ly 150cm by adding 2 0g of agarose to 100ml of TAE 1X buffer Mark the liquid level on the container then boil or 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 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 Prepare samples by mixing 10pl of each amplified sample with 2 5pl of 5X loading solution 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 prepared in Step 3 Set the voltage at 5 volts cm measured as the distance between the two electrodes Allow the gel to run for 2 hours After electrophoresis stain the gel in TAE 1X buffer containing 0 5pg 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 Photograph the gel using a UV transilluminator 302nm Note When analyzing the data do not be alarmed by extra bands in addition to the alleles DNA heteroduplexes can be expected when performing nondenaturing agarose gel electrophoresis The
48. mp PCR System 9600 9700 or 2400 thermal cyclers use 0 2ml MicroAmp reaction tubes For the Perkin Elmer model 480 thermal cycler we recommend 0 5ml GeneAmp thin walled reaction tubes In the order listed add the final volume of each reagent listed in Table 2 into a sterile 1 5ml amber colored tube Mix gently Table 2 PCR Master Mix for the PowerPlex 2 1 System VolumePer Numberof Final PCR Master Mix Component Reaction Reactions Volume nuclease free water 17 05p1 Gold ST R 10X Buffer 2 51 PowerPlex 2 1 10X Primer Pair Mix 2 5pl AmpliTaq Gold DNA polymerase 0 45gl 2 25u total volume 225pl T1 Add nuclease free water to the PCR master mix first then add Gold ST R 10X Buffer PowerPlex 2 1 10X Primer Pair Mix and AmpliTaq Gold DNA polymerase The template DNA will be added at Step 6 Assumes the AmpliTaq Gold DNA polymerase is at 5u yl If the enzyme concentration is different the volume of enzyme must be adjusted accordingly Note If the volume of AmpliTaq Gold DNA polymerase added to the master mix is less than 0 5pl you may 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 22 511 Do not store diluted AmpliTaq Gold DNA polymerase Pipet 22 5pl of PCR master mix into each reaction tube and place at room temperature Promega Corporatio
49. n 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Partt TMD011 Page 6 Printed in USA Revised 7 08 6 Pipet25yl of template DNA 1 2ng for each sample into the respective C tube containing 22 5p1 of PCR master mix e Note If the template DNA is stored in TE buffer the volume of DNA Promega added should not exceed 20 of the final reaction volume 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 may be present at low concentrations depending on the source of the template DNA and extraction procedure used 7 Forthe positive amplification control dilute K562 DNA to 0 4 0 8ng ul Pipet 2 5pl 1 2ng of diluted K562 DNA into a reaction tube containing 22 5pl of PCR master mix 8 For the negative amplification control pipet 2 5pl of nuclease free water instead of template DNA into a reaction tube containing 22 5pl of PCR master mix 9 If using the GeneAmp PCR System 9600 9700 or 2400 thermal cycler and MicroAmp reaction tubes or plates no addition of mineral oil to the reaction tubes is required However if using the model 480 thermal cycler and GeneAmp reaction tubes add one drop of mineral oil to each tube before closing Note Allow the mineral oil to flow down the side of the
50. n Asian American PowerPlex 1 2 System 0 9982125 0 9968853 0 9973337 0 9981793 PowerPlex 16 System 09999219 0 9999242 0 9997134 00 9999759 PowerPlex 1 1 System plus PowerPlex 2 1 System 14 STR loci 0 9999988 0 9999982 0 999995 0 9990995 PowerPlex 1 1 System System plus PowerPlex 2 1 System plus FFFL Muliplex 18 STR loci 0 9999999 00 9999999 09999095 09999999 9 E Methods for Polyacrylamide Gel Reuse A simple technique has been developed to allow reuse of gels while effectively eliminating the previous samples from the gel 12 With this technique a gel has been successfully reused up to eight times over a period of several days With the exception of the first and last lanes the second and third runs are as good as if not better than the first run The bands continue to remain sharp on subsequent runs but edge effects frowning of the outside lanes become progressively worse and ultimately limit gel reuse if more than 3 4 of the gel is to be analyzed Gel sizes 17cm x 43cm x 0 4cm 17cm x 32cm x 0 4cm Combs 15 2cm 34 well flat bottom 16 3cm 30 well flat bottom Electrophoresis apparatus Life Technologies Model SA EC600 power supply E C Apparatus Corporation Electrophoretic conditions Maintain a plate temperature of 45 to 50 C 60V cm at a constant power of 55W for 43cm gels Analysis Hitachi FMBIO II Fluorescence Imaging System Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399
51. otocol Product Size Cat PowerPlex 2 1 System 400 reactions DC6470 Not For Medical Diagnostic Use Cat DC6470 contains sufficient reagents for 400 reactions of 25pl each Includes Pre amplification Components Box Blue Label 4 x 300ul Gold STXR 10X Buffer 4 x 250l PowerPlex 2 1 10X Primer Pair Mix 3ug K562 DNA High Molecular Weight 10ng pl Postamplification Components Box Beige Label 4x150pl PowerPlex 2 1 Allelic Ladder Mix 4x 150ul Internal Lane Standard ILS 600 2x1ml Bromophenol Blue Loading Solution 250g Gel Tracking Dye 1 Protocol The PowerPlex 2 1 Allelic Ladder Mix is provided in a separate sealed bag for shipping This component should be moved to the postamplification box after opening Storage Conditions Store all components at 20 C in a nonfrost free freezer The PowerPlex 2 1 10X Primer Pair Mix PowerPlex 2 1 Allelic Ladder Mix and Internal Lane Standard 600 are light sensitive and must be stored in the dark The postamplification components are packaged separately to prevent cross contamination We strongly recommend that pre amplification and postamplification reagents be stored and used separately with different pipettes tube racks etc Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD011 Revised 7 08 Page 3 Product Components and Storage Condit
52. plate 10 5 2 1 0 5 0 2 or 0 1ng was amplified Results are shown in lanes 1 7 respectively Lane 8 shows amplification results with no DNA template Lanes labeled L contain allelic ladders for each of the nine loci Panel A A scan using a 505nm filter shows the fluorescein labeled loci Penta E D18551 D21S11 TH01 and D351358 Panel B A scan using a 585nm filter shows the TMR labeled loci FGA TPOX D851179 and vWA All materials were separated using a 5 denaturing Long Ranger polyacrylamide gel and detected using the Hitachi FMBIO II Fluorescence Imaging System A lane trace of lane 4 1ng template is shown to the right of each panel Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD011 Printed in USA Page 18 Revised 7 08 2654TA A 505nm Scan Fluorescein B 585nm Scan TMR L K562 L K562 Promega Penta E FGA D18551 EN TPOX D8S1179 32151 1 VWA THO1 D351358 oa 2655TA Figure 5 K562 DNA amplified using the PowerPlex 2 1 System K562 DNA 1ng was amplified Panel A A scan using a 505nm filter shows the fluorescein labeled loci Penta E D18551 D21511 TH01 and D3S1358 Panel B A scan using a 585nm filter shows the TMR labeled loci FGA TPOX D851179 and vWA All materials were separated using a 5 denaturing Long Ranger polyacrylamide gel and detected using the Hitachi FM
53. program High salt concentration or altered pH If the DNA template is stored in TE buffer that is not pH 8 0 or contains a higher EDTA concentration the DNA volume should not exceed 20 of the total reaction volume Carryover of K Nat Mg or EDTA from the DNA sample can negatively affect PCR A change in pH may also affect PCR Store DNA in TE buffer 10mM Tris HCl pH 8 0 0 1mM EDTA or nuclease free water Thermal cycler or tube problems Review the thermal cycling protocols in Section 4 B We have not tested other reaction tubes or thermal cyclers Calibrate the thermal cycler heating block if necessary Primer concentration was too low Use the recommended primer concentration Mix the 10X PowerPlex 2 1 Primer Pair for 15 seconds using a vortex mixer before use Samples were not completely denatured Heat denature samples at 95 C for 2 minutes and immediately chill on crushed ice or in an ice water bath prior to loading Poor quality polyacrylamide gel Prepare acrylamide and buffer solutions using high quality reagents We strongly recommend 5 Long Ranger acrylamide Cambrex Electrophoresis temperature was too high Run gel at lower temperature 40 50 C Following amplification lengthen the final extension step from 30 minutes at 60 C to 45 minutes n 1 bands may be generated when more than 1ng template DNA is used This is most commonly observed with the vWA and D391358 amplification products Reduce the amount of temp
54. rate protocols for use of the PowerPlex 2 1 System with the Perkin Elmer model 480 and GeneAmp PCR system 9600 9700 and 2400 thermal cyclers in addition to protocols for separation of amplified products and detection of separated material Protocols for operation of the fluorescence detection instruments should be obtained from the instrument manufacturer Information on other Promega fluorescent STR systems is available upon request from Promega or online at www promega com For information about detecting amplified STR fragments using silver staining 9 refer to the GenePrint STR Systems Technical Manual TMD004 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Partt TMD011 Page 2 Printed in USA Revised 7 08 2 Product Components and Storage Conditions C v Product Size Cat PowerPlex 2 1 System 100 reactions DC6471 Promega Not For Medical Diagnostic Use Cat DC6471 contains sufficient reagents for 100 reactions of 25pl each Includes Pre amplification Components Box Blue Label 1x 300pl1 Gold STXR 10X Buffer 1x 250p1 PowerPlex 2 1 10X Primer Pair Mix 9ug K562 DNA High Molecular Weight 10ng pl Postamplification Components Box Beige Label 1x150gd PowerPlex 2 1 Allelic Ladder Mix 1x 150pl Internal Lane Standard ILS 600 1x1ml Bromophenol Blue Loading Solution 250g Gel Tracking Dye 1 Pr
55. ry In The Third International Symposium on Human Identification 1992 Promega Corporation 245 69 11 Hartmann J M et al 1991 Guidelines for a quality assurance program for DNA analysis Crime Laboratory Digest 18 44 75 12 Tereba A Micka K A and Schumm J W 1998 Reuse of denaturing polyacrylamide gels for short tandem repeat analysis BioTechniques 25 892 7 13 Budowle B et al 1991 Analysis of the VNTR locus D1580 by the PCR followed by high resolution PAGE Am J Hum Genet 48 137 44 14 Nakamura Y et al 1987 Variable number of tandem repeat VNTR markers for human gene mapping Science 235 1616 22 15 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 16 Levinson G and Gutman G A 1987 Slipped strand mispairing A major mechanism for DNA sequence evolution Mol Biol Evol 4 203 21 17 Schlotterer C and Tautz D 1992 Slippage synthesis of simple sequence DNA Nucleic Acids Res 20 211 5 18 Smith J R et al 1995 Approach to genotyping errors caused by nontemplated nucleotide addition by Tag DNA polymerase Genome Res 5 312 7 19 Magnuson V L et al 1996 Substrate nucleotide determined non templated addition of adenine by Taq DNA polymerase Implications for PCR based genotyping BioTechniques 21 700 9 20 Walsh P S Fildes N J and Reynolds R 1996
56. ssues Note If using a squaretooth comb the shorter glass plate requires bind silane treatment see below The plates do not require a special silane treatment when using a sharkstooth comb Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD011 Revised 7 08 Page 9 Promega 5 A Polyacrylamide Gel Preparation continued Bind Silane Treatment of Glass Plate Prepare fresh binding solution in a chemical fume hood Add 1pl of bind silane to a 1 5ml microcentrifuge tube containing 0 5ml of 0 5 acetic acid in 95 ethanol Wipe the etched side of the shorter glass plate in the comb region using a Kimwipes tissue saturated with freshly prepared binding solution Wait 5 minutes for the binding solution to dry Wipe the shorter glass plate 3 4 times with 95 ethanol and Kimwipes tissues in the comb area to remove excess binding solution Assemble the glass plates by placing 0 4mm side spacers between the front and rear glass plates using clamps to hold them in place 3 4 clamps on each side A bottom spacer is neither required nor recommended Place the assembly horizontally on a test tube rack or similar support Prepare a 5 Long Ranger acrylamide solution by combining the ingredients listed in Table 3 for Long Ranger acrylamide gels Table 3 Preparation of 5 Long Ranger Polyacrylamide
57. tachi FMBIOS II Fluorescence Imaging System Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD011 Printed in USA Page 16 Revised 7 08 2652TA Internal Lane Standard 600 C e 200 180 160 140 120 100 2653TA Figure 3 The Internal Lane Standard 600 The Internal Lane Standard 600 contains fragments ranging from 60 to 600 bases in length It was mixed with the samples shown in Figure 2 before loading the gel Following separation the Internal Lane Standard 600 was detected using a 650nm filter with the Hitachi FMBIOS II Fluorescence Imaging System Fragments smaller than 100 bases are not shown on this gel Fragment sizes are shown to the right of the gel The 100 200 300 400 500 and 600 base fragments display double the intensity of the others Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Partt TMD011 Revised 7 08 Page 17 C A 505nm Scan Fluorescein B 585nm Scan TMR eo L12345678L L120 456791 Promega TEEEEE UT EEUN Penta E FGA Seoo D18551 M TPOX BGD D8S1179 D21911 BREE THO PER VWA ae m o D391358 kofra Figure 4 Amplification of various amounts of template with the PowerPlex 2 1 System A single DNA tem
58. te is occasionally updated and has numerous links to many other useful sites Additional STR references also 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 Phone 608 274 4330 Fax 608 277 2516 www promega com Partt TMD011 Page 24 Printed in USA Revised 7 08 9 Appendix C s 9 A Advantages of STR Typing STR typing is more tolerant of degraded DNA templates than other typing methods because amplification products are less than 500bp long much smaller than material detected using AMP FLP 13 or VNTR 14 analysis STR typing is also amenable to a variety of rapid DNA purification techniques that are compatible with PCR but do not provide enough DNA of appropriate quality for Southern blot based analyses Amplification products generated with Promega STR products are generally of discrete and separable lengths This allows construction of allelic ladders containing fragments of the same lengths as several or all known alleles for each locus Visual or software based comparison between the allelic ladder and amplified samples of the same locus allows rapid and precise assignment of alleles Results obtained using the PowerPlex 2 1 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 15
59. tems e Nuclease Free Water Cat P1193 e Mineral Oil Cat DY1151 for use with the model 480 thermal cycler We routinely amplify 1 2ng of template DNA in a 25pl reaction volume using the protocols detailed below Preferential amplification of smaller loci can occur Expect to see more intense bands for smaller loci and relatively less intense bands for larger loci if more than the recommended amount of template is used Reduce the amount of template DNA or the number of cycles to correct this Primer concentrations are optimized for use with the GeneAmp PCR systems 9600 thermal cycler and AmpliTaq Gold DNA polymerase as described in the amplification protocol provided below Thermal cycling conditions are also provided for the GeneAmp PCR system 9700 and 2400 and Perkin Elmer model 480 thermal cyclers Use of AmpliTaq Gold DNA polymerase is always recommended DNA template of 5ng or greater results in an imbalance in more intense bands from locus to locus The smaller loci show greater amplification yield than larger loci Reduce the number of cycles in the amplification program by 2 or 4 cycles i e 10 18 or 10 16 cycling to improve locus to locus balance 4 4 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 set
60. tube and form an overlay to limit sample loss or cross contamination due to splattering 10 Centrifuge samples briefly to contents to the bottom of the tube 4 B Amplification Thermal Cycling This manual contains protocols for use of the PowerPlex 2 1 System with the Perkin Elmer model 480 and GeneAmp PCR system 9600 9700 and 2400 thermal cyclers For information about other thermal cyclers please contact Promega Technical Services by e mail genetic promega com 1 Place tubes in the thermal cycler 2 Select and run a recommended protocol The preferred protocols for use with the GeneAmp PCR System 9600 9700 and 2400 thermal cyclers and Perkin Elmer model 480 thermal cycler are provided below Note Ramp settings displayed in the cycling profile indicate the ramp to the temperature that follows 3 After completion of the thermal cycling protocol store samples at 20 C in a light protected box Note Storage of amplified samples at 4 C or higher may produce degradation products Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD011 Revised 7 08 Page 7 Protocol for the GeneAmp PCR System 9700 Thermal Cycler 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 t
61. up Use equipment and supplies dedicated for amplification setup Meticulous care must be taken to ensure successful amplification A guide to amplification troubleshooting is provided in Section 7 1 Thaw the Gold STXR 10X Buffer and PowerPlex 2 1 10X Primer Pair Mix Notes 1 Mix reagents by vortexing for 15 seconds before each use Do not centrifuge the 10X Primer Pair Mix as this may cause the primers to be concentrated at the bottom of the tube 2 A precipitate may form in the Gold ST R 10X Buffer If this occurs warm the solution briefly at 37 C then vortex until the precipitate is in solution Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD011 Revised 7 08 Page 5 Promega 4 A Amplification Setup continued 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 compensate 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 Place one clean 0 2ml or 0 5ml reaction tube for each reaction into a rack and label appropriately Alternatively use a MicroAmp plate and label appropriately Note If using the GeneA
62. ve 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 Phone 608 274 4330 Fax 608 277 2516 www promega com Partt TMDO11 Page 38 Printed in USA Revised 7 08
63. ye is loaded in the outermost lane of the gel at least three lanes from the nearest sample and is used as a visual indicator of migration Briefly centrifuge samples to bring contents to the bottom of the tubes Denature samples by heating at 95 C for 2 minutes and immediately chill on crushed ice or in an ice water bath Denature samples just prior to loading the gel Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Partt TMD011 Page 12 Printed in USA Revised 7 08 5 After the pre run use a 50 100cc syringe filled with buffer to flush urea from the well area If using a squaretooth comb do not reinsert the comb V as the samples will be loaded directly into the wells If using a sharkstooth Pro ega comb insert the teeth into the gel approximately 1 2mm and leave the m comb inserted in the gel during both gel loading and electrophoresis 6 Load3pl of each denatured sample into the respective wells The loading process should take no longer than 20 minutes to prevent the gel from cooling 5 D Gel Electrophoresis 1 After loading run the gel using the same conditions as in Section 5 B Observe the lane containing Gel Tracking Dye to monitor sample migration In a 5 Long Ranger acrylamide gel xylene cyanol dye migrates at approximately 190 bases and bromophenol blue dye migrates at less than 80 bases 2

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