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PowerPlex® 16 HS System

1. 31 H Results 32 7 Troubleshooting 34 A Amplification and Fragment Detection 34 B GeneMapper ID Analysis Software 37 C PowerTyper 16 Macro 40 8 References 41 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 TMD022 1 09 PowerPlex 16 HS System 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 Page 2 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in U
2. 100 reactions DC6651 PowerPlex ES Monoplex System SE33 JOE 100 reactions DC6751 PowerPlex 1 2 System 100 reactions DC6101 PowerPlex 16 BIO System 100 reactions DC6541 400 reactions DC6540 PowerPlex ES System 100 reactions DC6731 400 reactions DC6730 PowerPlex Y System 50 reactions DC6761 200 reactions DC6760 Not for Medical Diagnostic Use Page 51 Accessory Components Product Size Cat PowerPlex Matrix Standards 310 50 l each dye DG4640 PowerPlex Matrix Standards 3100 3130 25 l each dye DG4650 PowerTyper Macros 1 CD ROM DG3470 Internal Lane Standard 600 150 l DG1071 Mineral Oil 12ml DY1151 Water Amplification Grade 5 1 250 l DW0991 Not for Medical Diagnostic Use For Laboratory Use Sample Preparation Systems Product Size Cat DNA IQ System 100 reactions DC6701 400 reactions DC6700 Differex System 50 samples DC6801 200 samples DC6800 Maxwell 16 Instrument each AS2000 DNA IQ Reference Sample Kit for Maxwell 16 48 preps AS1040 DNA IQ Casework Sample Kit for Maxwell 16 48 preps AS1210 Plexor HY System 800 reactions DC1000 200 reactions DC1001 Slicprep 96 Device 10 pack V1391 Not for Medical Diagnostic Use For Laboratory Use For Research Use Only Not for use in diagnostic procedures ART Aerosol Resistant Tips Product Volume Size ti
3. Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD022 Printed in USA 1 09 6 H Results Representative results of the PowerPlex 16 HS System are shown in Figure 9 The PowerPlex 16 HS Allelic Ladder Mix is shown in Figure 10 The PowerPlex 16 Monoplex System Penta E Fluorescein Cat DC6591 and PowerPlex 16 Monoplex System Penta D JOE Cat DC6651 are available to amplify the Penta E and Penta D loci respectively These monoplex systems allow amplification of a single locus to confirm results obtained with the PowerPlex 16 System PowerPlex 16 HS System PowerPlex 16 BIO System or PowerPlex 2 1 System Figure 9 The PowerPlex 16 HS System A single template DNA 0 5ng was amplified using the PowerPlex 16 HS 10X Primer Pair Mix Amplification products were mixed with Internal Lane Standard 600 and analyzed with an Applied Biosystems 3130 Genetic Analyzer using a 3kV 5 second injection Results were analyzed using GeneMapper ID software version 3 2 Panel A An electropherogram showing the peaks of the fluorescein labeled loci D3S1358 TH01 D21S11 D18S51 and Penta E Panel B An electropherogram showing the peaks of the JOE labeled loci D5S818 D13S317 D7S820 D16S539 CSF1PO and Penta D Panel C An electropherogram showing the peaks of the TMR labeled loci Amelogenin vWA D8S1179 TPOX and FGA Panel D An electropherogram
4. Operating Systems GeneScan Software and Macintosh Operating Systems ABI PRISM 3100 or 3100 Avant Genetic Analyzer with Data Collection Software Version 2 0 Section 5 A ABI PRISM 3100 Genetic Analyzer with Data Collection Software Version 1 0 1 or 1 1 Section 5 B ABI PRISM 310 Genetic Analyzer Section 5 C Figure 1 An overview of the PowerPlex 16 HS System protocol Page 4 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 TMD022 Printed in USA 1 09 2 Product Components and Storage Conditions Product Size Cat PowerPlex 16 HS System 100 reactions DC2101 Not For Medical Diagnostic Use This system contains sufficient reagents for 100 reactions of 25 l each Includes Pre amplification Components Box Blue Label 1 500 l PowerPlex HS 5X Master Mix 1 250 l PowerPlex 16 HS 10X Primer Pair Mix 25 l 9947A DNA 10ng l 5 1 250 l Water Amplification Grade Post amplification Components Box Beige Label 1 25 l PowerPlex 16 HS Allelic Ladder Mix 1 150 l Internal Lane Standard ILS 600 Product Size Cat PowerPlex 16 HS System 400 reactions DC2100 Not For Medical Diagnostic Use This system contains sufficient reagents for 400 reactions of 25 l each Includes Pre amplification Components Box Blue Label
5. The analysis parameters can be saved in the Params folder in most installations this is located at C AppliedBio Shared Analysis Sizecaller Params 5 Apply the stored analysis parameters file to the samples 6 Assign a new size standard Select a sample file and highlight the arrow next to size standard Select define new Assign the size standard peaks as shown in Figure 13 in Section 9 E Store the size standard in the Size Standards folder at C AppliedBio Shared Analysis Sizecaller SizeStandards 7 Apply the size standard file to the samples then analyze the sample files See Section 6 F for additional information on the use of the PowerTyper 16 Macro Release 2 0 and Genotyper software Notes 1 Peak heights outside the linear range of the instrument may generate artifact peaks due to instrument saturation i e overloading the sample Bleedthrough pull ups from one color to another may be observed Saturated signal may also appear as two peaks split peak 2 If peak heights are not within the linear range of detection of the instrument the ratio of stutter peaks to real allele peaks increases and allele designations become difficult to interpret The balance of peak heights may also appear less uniform 3 There can be variation between instruments regarding the relative fluorescence levels detected using the same sample Furthermore different instruments vary in the relative efficiency of color
6. 4 500 l PowerPlex HS 5X Master Mix 4 250 l PowerPlex 16 HS 10X Primer Pair Mix 25 l 9947A DNA 10ng l 10 1 250 l Water Amplification Grade Post amplification Components Box Beige Label 4 25 l PowerPlex 16 HS Allelic Ladder Mix 4 150 l Internal Lane Standard ILS 600 The PowerPlex 16 HS Allelic Ladder Mix is provided in a separate sealed bag for shipping This component should be moved to the post amplification box after opening Storage Conditions Store all components at 20 C in a nonfrost free freezer The PowerPlex 16 HS 10X Primer Pair Mix PowerPlex 16 HS Allelic Ladder Mix and Internal Lane Standard 600 are light sensitive and must be stored in the dark We strongly recommend that pre amplification and post amplification reagents be stored and used separately with different pipettes tube racks etc For daily use the PowerPlex 16 HS 10X Primer Pair Mix and PowerPlex HS 5X Master Mix can be stored at 4 C for up to a total of 1 week without loss of activity The 9947A DNA and Water Amplification Grade can be stored at 4 C long term Page 5 Available Separately Product Size Cat PowerTyper Macros Release 2 0 1 CD ROM DG3470 Not For Medical Diagnostic Use The PowerTyper Macros Release 2 0 for use with Genotyper software are available from Promega This CD ROM contains the file PowerTyper 16 Macro Release 2 0 for use with the
7. 691195 and other patents pending c U S Pat Nos 5 843 660 6 479 235 6 221 598 and 7 008 771 Australian Pat No 724531 Chinese Pat No 10366753 Canadian Pat No 2 118 048 Korean Pat No 290332 Singapore Pat No 57050 Japanese Pat No 3602142 European Pat No 0960207 and other patents pending d Licensed under U S Pat Nos 5 338 671 and 5 587 287 and corresponding patents in other countries e Allele sequences for one or more of the loci vWA FGA D8S1179 D21S11 and D18S51 in allelic ladder mixtures is licensed under U S Pat No 7 087 380 Australia Pat No 2003200444 and corresponding patent claims outside the US 2009 Promega Corporation All Rights Reserved Maxwell Plexor and PowerPlex are registered trademarks of Promega Corporation Differex DNA IQ PowerTyper and Slicprep are trademarks of Promega Corporation ABI PRISM GeneMapper GeneScan Genotyper and MicroAmp are registered trademarks of Applera Corporation ART is a registered trademark of Molecular Bio Products Inc Biomek is a registered trademark of Beckman Coulter Inc 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 GeneAmp is a registered trademark of Roche Molecular Systems Inc Hi Di and POP 4 are trademarks of Applera Corporation Macintosh is a regi
8. Alleles of STR loci are differentiated 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 16 HS System a e allows co amplification and three color detection of sixteen loci fifteen STR loci and Amelogenin including Penta E D18S51 D21S11 TH01 D3S1358 FGA TPOX D8S1179 vWA Amelogenin Penta D CSF1PO D16S539 D7S820 D13S317 and D5S818 One primer for each of the Penta E D18S51 D21S11 TH01 and D3S1358 loci is labeled with fluorescein FL one primer for each of the FGA TPOX D8S1179 vWA and Amelogenin loci is labeled with carboxy tetramethylrhodamine TMR and one primer for each of the Penta D CSF1PO D16S539 D7S820 D13S317 and D5S818 loci is labeled with 6 carboxy 4 5 dichloro 2 7 dimethoxy fluorescein JOE All sixteen loci are amplified simultaneously in a single tube and analyzed in a single injection or gel lane The PowerPlex 16 HS System is compatible with the ABI PRISM 310 3100 and 3100 Avant Genetic Analyzers and Applied Biosystems 3130 and 3130xl Genetic Analyzers The protocols presented in this manual were tested at Promega Corporation Amplification and detection instrumentation may vary You may need to optimize protocols including cycle number and injection time or loading volume for each laboratory instrument In
9. B Moller A and Wiegand P 1995 Structure of new mutations in 2 STR systems Int J Leg Med 107 201 3 24 Griffiths R et al 1998 New reference allelic ladders to improve allelic designation in a multiplex STR system Int J Legal Med 111 267 72 25 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 26 Gill P et al 1997 Considerations from the European DNA Profiling Group EDNAP concerning STR nomenclature Forensic Sci Int 87 185 92 27 Fr geau C J et al 1995 Characterization of human lymphoid cell lines GM9947 and GM9948 as intra and interlaboratory reference standards for DNA typing Genomics 28 184 97 28 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 29 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 30 Puers C et al 1993 Identification of repeat sequence heterogeneity at the polymorphic STR locus HUMTH01 AATG n and reassignment of alleles in population analysis using a locus specific allelic ladder Am J Hum Genet 53 95
10. New 4 Select Basic or Advanced Figure 5 The type of analysis method selected must match the type of analysis method created earlier Select OK 5 Enter a detailed name such as ILS 600 advanced in the Size Standard Editor Figure 6 6 Choose red as the color for the size standard dye 7 Enter the sizes of the internal lane standard fragments see Section 9 E Figure 13 8 Select OK Page 25 Processing Data for Databasing or Paternity Samples 1 Import sample files into a new project as described in the Applied Biosystems GeneMapper ID Software Human Identification Analysis Tutorial 2 In the Sample Type column use the drop down menu to select Ladder Sample Positive Control or Negative Control Every folder in the project must contain at least one ladder that is designated as such for proper genotyping 3 In the Analysis Method column select the analysis method created in the Creating a Databasing or Paternity Analysis Method section 4 In the Panel column select PowerPlex_16_ID3 2 X where X refers to the most recent version of the panel files This is the panel set that was imported in Section 6 A 5 In the Size Standard column select the size standard that was created in the Creating a Size Standard section 6 If analyzing data from an ABI PRISM 310 Genetic Analyzer ensure that the appropriate matrix file is selected in t
11. PCR Amplification Mix Component Volume Per Reaction Number of Reactions Final Volume l PowerPlex HS 5X Master Mix 5 0 l PowerPlex 16 HS 10X Primer Pair Mix 2 5 l Water Amplification Grade1 l Per tube template DNA volume1 0 25 1ng up to 17 5 l total reaction volume 25 l 1The PCR amplification mix volume and template DNA volume should total 25 l Consider the volume of template DNA and add Water Amplification Grade to the PCR amplification mix to bring the final volume of the final reaction to 25 l Page 44 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 TMD022 Printed in USA 1 09 9 B 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 14 or VNTR 15 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
12. etc until all ladders in the project are used If an allelic ladder fails to be analyzed or if many off ladder alleles are found in the samples samples should be re analyzed using another ladder from the project 7 Double click on the Display Fluorescein Data macro to display the blue dye for all sample injections lanes Scroll down to observe and edit as needed 8 Double click on the Display TMR Data macro to display the yellow dye for all sample injections lanes Scroll down to observe and edit as needed 9 Double click on the Display JOE Data macro to display the green dye for all sample injections lanes Scroll down to observe and edit as needed Page 31 10 Create the appropriate table by selecting the PowerTable Make Allele Table or Make CODIS Table macro The three available table formats are shown below The PowerTable option allows up to four alleles per sample file Additional information such as low peak signal or high peak signal is also included The Allele Table and CODIS Table options include only two alleles per locus If more than two alleles are present at a locus the smallest alleles identified are included The Allele Table format displays the categories loci in columns while the CODIS table format displays the categories in rows These tables can be customized to fit needs To save data in tables go to the Table drop down menu highlight Export to File and save the file with the desired name and lo
13. showing the 60bp to 500bp fragments of the Internal Lane Standard 600 A B C D 7918TA Page 33 Artifacts and Stutter Stutter bands are a common amplification artifact associated with STR analysis Stutter products are often observed one repeat unit below the true allele peak and occasionally two repeat units smaller or one repeat unit larger than the true allele peak Frequently alleles with a greater number of repeat units will exhibit a higher percent stutter The pattern and intensity of stutter may differ slightly between primer sets for the same loci In addition to stutter peaks other artifact peaks can be observed at some of the PowerPlex 16 HS System loci Low level products can be seen in the n 2 and n 2 positions two bases below and above the true allele peak respectively with some loci such as D21S11 Samples may show low level artifacts in the regions between D21S11 and D18S51 D7S820 and D16S539 and D8S1179 and TPOX Occasionally an off ladder artifact can be seen in the 690 691bp position in the fluorescein dye channel One or more extra peaks that are not directly related to amplification may be observed in the D3S1358 TH01 D21S11 and Penta E region of the fluorescein channel D13S317 and D16S539 region of the JOE channel and vWA region of the TMR channel These extra peaks occur when the amplified peaks are particularly intense high signal level or template amount formamide polymer or capi
14. 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 TMD022 Printed in USA 1 09 6 C Creating a Databasing or Paternity Analysis Method with GeneMapper ID Software 1 Select Tools then GeneMapper Manager 2 Select the Analysis Methods tab 3 Select New and a new analysis method dialog box will open 4 Select HID and select OK Note If you do not see the HID option you do not have the GeneMapper ID software Contact Promega Technical Services by e mail genetic promega com for assistance 5 Enter a descriptive name for the analysis method such as PowerPlex16_20 filter 6 Select the Allele tab 7 Select the bin set corresponding to the PowerPlex System Promega_Bins_ID3 2 X where X refers to the most recent version of the bin set 8 Ensure that the Use marker specific stutter ratio if available box is checked 9 Enter the values shown in Figure 7 for proper filtering of peaks when using the PowerPlex 16 HS System For an explanation of the proper usage and effect of these settings refer to the Applied Biosystems user bulletin titled Installation Procedures and New Features for GeneMapper ID Software 3 2 Creating a Size Standard 1 Select Tools then GeneMapper Manager 2 Select the Size Standard tab 3 Select
15. C for 3 minutes and immediately chill on crushed ice or in an ice water bath for 3 minutes Denature samples just prior to loading 7 Assemble the tubes in the appropriate autosampler tray 48 or 96 tube 8 Place the autosampler tray in the instrument and close the instrument doors Instrument Preparation Refer to the instrument users manual for instructions on cleaning the pump block installing the capillary calibrating the autosampler and adding polymer to the syringe 1 Open the ABI PRISM 310 data collection software 2 Prepare a GeneScan sample sheet as described in the ABI PRISM 310 Genetic Analyzer User s Manual Enter the appropriate sample information in the sample info column For rows containing PowerPlex 16 HS Allelic Ladder Mix insert the word ladder in the sample info column for the blue dye color yellow dye color and green dye color This information must be entered to successfully analyze your data using the PowerTyper 16 Macro Release 2 0 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 TMD022 1 09 Page 18 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 TMD022 Printed in USA 1 09 5 C Detection of Am
16. PowerPlex 16 HS System The macros can be also downloaded at www promega com geneticidtools The proper panel and bin files for use with GeneMapper ID software can be obtained from the Promega web site at www promega com geneticidtools panels_bins Matrix standards are required for initial setup of the color separation matrix The matrix standards are sold separately and are available for the ABI PRISM 310 Genetic Analyzer PowerPlex Matrix Standards 310 Cat DG4640 and the ABI PRISM 3100 and 3100 Avant Genetic Analyzers and Applied Biosystems 3130 and 3130xl Genetic Analyzers PowerPlex Matrix Standards 3100 3130 Cat DG4650 See Section 9 H for ordering information 3 Before You Begin 3 A 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 10 11 Guidelines for the validation process are published in the Internal Validation of STR Systems 12 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 and fluorescence detection PCR based STR analysis is subject to contamination by very small amounts of human DNA Extreme care should be taken to avoid cross contamination when preparing s
17. Proper generation of a matrix file is critical to evaluate multicolor systems with the ABI PRISM 310 3100 and 3100 Avant Genetic Analyzers and Applied Biosystems 3130 and 3130xl Genetic Analyzers A matrix must be generated for each individual instrument The PowerPlex Matrix Standards 310 Cat DG4640 is required for matrix standardization for the ABI PRISM 310 Genetic Analyzer For best results the PowerPlex Matrix Standards 3100 3130 Cat DG4650 should not be used to generate a matrix on the ABI PRISM 310 Genetic Analyzer The PowerPlex Matrix Standards 3100 3130 Cat DG4650 is required for spectral calibration on the ABI PRISM 3100 and 3100 Avant Genetic Analyzers and Applied Biosystems 3130 and 3130xl Genetic Analyzers The PowerPlex Matrix Standards 310 Cat DG4640 cannot be used to generate a matrix on these instruments For protocols and additional information on matrix standardization see the PowerPlex Matrix Standards 310 Technical Bulletin TBD021 For protocols and additional information on spectral calibration see the PowerPlex Matrix Standards 3100 3130 Technical Bulletin TBD022 These manuals are available online at www promega com tbs 4 Protocols for DNA Amplification Using the PowerPlex 16 HS System Materials to Be Supplied by the User PerkinElmer model 480 or GeneAmp PCR System 9600 or 9700 thermal cycler Applied Biosystems microcentrifuge 0 5ml GeneA
18. fibrinogen alpha chain gene TTTC Complex 24 TPOX TMR 2p24 2pter HUMTPOX human thyroid peroxidase gene AATG D8S1179 TMR 8q NA TCTA Complex 24 vWA TMR 12p12 pter HUMVWFA31 human von Willebrand factor gene TCTA Complex 24 Amelogenin2 TMR Xp22 1 22 3 and Y HUMAMEL human Y chromosomal gene for Amelogenin like protein NA Penta D JOE 21q NA AAAGA CSF1PO JOE 5q33 3 34 HUMCSF1PO human c fms proto oncogene for CSF 1 receptor gene AGAT D16S539 JOE 16q24 qter NA GATA D7S820 JOE 7q11 21 22 NA GATA D13S317 JOE 13q22 q31 NA TATC D5S818 JOE 5q23 3 32 NA AGAT 1The August 1997 report 25 26 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 2Amelogenin 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 106 base X specific band TMR carboxy tetramethylrhodamine FL fluorescein JOE 6 carboxy 4 5 dichloro 2 7 dimethoxyfluorescein NA not applicable Page 46 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 U
19. for the 3100 Hi Di formamide Applied Biosystems Cat 4311320 PowerPlex Matrix Standards 3100 3130 Cat DG4650 The quality of formamide is critical Use Hi Di formamide with a conductivity less than 100 S cm Freeze formamide in aliquots at 20 C Multiple freeze thaw cycles or long term storage at 4 C may cause a breakdown of the formamide Formamide with a conductivity greater than 100 S cm may contain ions that compete with DNA during injection which results in lower peak heights and reduced sensitivity A longer injection time may not increase the signal Caution Formamide is an irritant and a teratogen 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 formamide Sample Preparation 1 Prepare a loading cocktail by combining and mixing Internal Lane Standard 600 and Hi Di formamide as follows 0 5 l ILS 600 injections 9 5 l Hi Di formamide injections Note The volume of internal lane standard used in the loading cocktail can be increased or decreased to adjust the intensity of the size standard peaks The optimal peak height for the 100 base fragment of the internal lane standard is 500 1 000RFU If peak heights are too low we recommend altering the formamide internal lane standard mix to contain 1 0 l of ILS 600 and 9 0 l of Hi Di for
20. heights can be high if the samples are overloaded See Section 6 H for additional information on stutter and artifacts Artifacts of STR amplification PCR amplification of STR systems can result in artifacts that appear as peaks 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 or decrease injection time See Section 5 CE related artifacts spikes Minor voltage changes or urea crystals passing by the laser can 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 samples to confirm Excessive amount of DNA Amplification of gt 1ng template can result in a higher number of stutter bands Use less template DNA or reduce the number of cycles in the amplification program by 2 4 cycles 10 20 or 10 18 cycling Pull up or bleedthrough Pull up can occur when peak heights are too high or if a poor or incorrect matrix has been applied to the samples For the ABI PRISM 310 Genetic Analyzer generate a new matrix and apply it to the samples For the ABI PRISM 3100 and 3100 Avant Genetic Analyzers and Applied Biosystems 3130 and 3130xl Genetic Analyzers perform a new spectral calibration and re run the samples Instrument sensitivities can vary Opti
21. house validation should be performed The PowerPlex 16 HS System provides all of the materials necessary for amplification of STR regions of purified genomic DNA including hot start Taq DNA polymerase This manual contains separate protocols for use of the PowerPlex 16 HS System with the Perkin Elmer model 480 and GeneAmp PCR system 9600 and 9700 thermal cyclers in addition to protocols to separate amplified products and detect separated material Figure 1 Protocols for operation of the fluorescence detection instruments should be obtained from the instrument manufacturer Information on other Promega fluorescent STR systems including the PowerPlex 16 Monoplex Systems and detection of amplified STR fragments using silver staining is available upon request from Promega or online at www promega com Page 3 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 TMD022 1 09 Amplification Setup Thermal Cycling Instrument Setup and Sample Preparation Data Analysis Section 4 B Section 5 Section 6 Section 4 A GeneAmp PCR System 9700 GeneAmp PCR System 9600 Model 480 Thermal Cycler Applied Biosystems 3130 or 3130xl Genetic Analyzer with Data Collection Software Version 3 0 Section 5 A GeneMapper ID Software Versions 3 1 and 3 2 GeneScan Software and Windows
22. in paternity analyses is the paternity index PI a means for presenting the genetic odds in favor of paternity given the genotypes for the mother child and alleged father 36 The typical paternity indices for the PowerPlex 16 HS System are shown in Table 6 The PowerPlex 16 HS System provides typical paternity indices exceeding 500 000 in each population group An alternative calculation used in paternity analyses is the power of exclusion 36 This value calculated for the PowerPlex 16 HS System exceeds 0 999998 in all populations tested Table 6 9 E The Internal Lane Standard 600 The Internal Lane Standard ILS 600 contains 22 DNA fragments of 60 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 Figure 13 Each fragment is labeled with carboxy X rhodamine CXR and can be detected separately as a fourth color in the presence of PowerPlex 16 HS amplified material The ILS 600 is designed for use in each gel lane or CE injection to increase precision in analyses when using the PowerPlex 16 HS System Protocols for preparation and use of this internal lane standard are provided in Section 5 Table 6 Matching Probabilities Paternity Indices and Power of Exclusion of the PowerPlex 16 HS System in Various Populations African American Caucasian American Hispanic American Asian American Matching Probability 1 in 1 41 1018 1 in 1 83 101
23. is not HID it should be deleted and a new analysis method created 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 TMD022 1 09 Page 39 Page 40 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 TMD022 Printed in USA 1 09 7 C PowerTyper 16 Macro Symptoms Causes and Comments File does not open Genotyper software was not installed Be certain that the on your computer Genotyper software version 2 5 Macintosh or version 3 6 or higher Windows NT is installed Incorrect version of Genotyper software The PowerTyper 16 Macro will not work with Genotyper software versions prior to version 2 5 The CD ROM may have been damaged during shipment Contact Technical Services by e mail genetic promega com The file was corrupted during download or transfer Download the file again or obtain the file on CD ROM Error message Allelic ladder sample files were not identified Be certain the Could not complete the sample info or color info column for each lane containing Run Macro command because PowerPlex 16 HS Allelic Ladder Mix contains the word no dye lanes are selected ladder The macro uses the word ladder to identify samp
24. occurs because different sequences in allelic ladder and ILS components may cause differences in migration The dye label also affects migration of alleles 3For a current list of microvariants see the Variant Allele Report published at the U S National Institute of Standards and Technology NIST web site at www cstl nist gov div831 strbase 4Amelogenin is not an STR but displays a 106 base X specific band and a 112 base Y specific band Page 47 Terminal nucleotide addition 19 20 occurs when Taq DNA polymerase adds a nucleotide generally adenine to the 3 ends of amplified DNA fragments in a template independent manner The efficiency with which this occurs varies with different primer sequences Thus an artifact band one base shorter than expected 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 21 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 22 23 Thus FGA and D21S11 display numerous relatively common microvariants For reas
25. 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 16 HS 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 16 9 C Advantages of Using the Loci in the PowerPlex 16 HS System The loci included in the PowerPlex 16 HS System Tables 3 and 4 have been selected because they satisfy the needs of several major standardization bodies throughout the world For example the United States Federal Bureau of Investigation FBI has selected 13 STR core loci for typing prior to searching or including submitting samples in CODIS Combined DNA Index System the U S national database of convicted offender profiles The PowerPlex 16 HS System amplifies all CODIS core loci in a single reaction The PowerPlex 16 HS System also contains two low stutter highly polymorphic pentanucleotide repeat loci Penta E and Penta D These additional loci add significantly to the discrimination power of the system making the PowerPlex 16 HS System a single amplification system with a power of exclusion sufficient to resolve paternity disputes definitively In addition the extremely low level of stutter seen with Penta E and Penta D makes them ideal
26. 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 on 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 genetic promega com 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 TMD022 1 09 Figure 13 Internal Lane Standard 600 An electropherogram showing Internal Lane Standard 600 fragments 5751TA 1 200 1 000 800 600 400 200 0 60 80 100 120 140 160 180 200 225 250 275 300 325 350 375 400 425 450 475 500 550 600 TE 4 buffer 10mM Tris HCl 0 1mM EDTA pH 8 0 2 21g Tris base 0 037g EDTA Na2EDTA 2H2O Dissolve Tris base and EDTA in 900ml of deionized water Adjust to pH 8 0 with HCl Bring the final volume to 1 liter with deionized water Page 50 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 TMD022 Printed in USA 1 09 9 G DNA Extraction and Quantitation Methods continued To process sexual assault samples differential extraction can be used t
27. 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 2 shows the appropriate 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 Figure 2 The ramp rates for the GeneAmp PCR System 9700 thermal cycler 7486MA 94 0 C 100 90 0 C 100 60 0 C 29 60 0 C 29 70 0 C 23 70 0 C 23 3 tmp 10 cycles 3 tmp 22 cycles Page 11 5 Instrument Setup and Sample Preparation 5 A Detection of Amplified Fragments Using the ABI PRISM 3100 or 3100 Avant Genetic Analyzer with Data Collection Software Version 2 0 and the Applied Biosystems 3130 or 3130xl Genetic Analyzer with Data Collection Software Version 3 0 Materials to Be Supplied by the User 95 C dry heating block water bath or thermal cycler crushed ice or ice water bath centrifuge compatible with 96 well plates aerosol resistant pipette tips 3100 or 3130 capillary array 36cm performance optimized polymer 4 POP 4 for the 3100 or 3130 10X genetic analyzer buffer with EDTA MicroAmp optical 96 well plate and septa Hi Di formamide Applied Biosystems Cat 4311320 PowerPlex Matrix Standar
28. the instrument Instrument Preparation Refer to the instrument users manual for instructions on cleaning installing the capillary array performing a spatial calibration and adding polymer Analyze samples as described in the user s manual for the ABI PRISM 3100 or 3100 Avant Genetic Analyzer with data collection software version 2 0 and the Applied Biosystems 3130 or 3130xl Genetic Analyzer with the following exceptions 1 In the Module Manager select New Select Regular in the Type drop down list and select HIDFragmentAnalysis36_POP4 in the Template drop down list Confirm that the injection time is 5 seconds and the injection voltage is 3kV Lengthen the run time to 1 800 seconds Give a descriptive name to your run module and select OK Note Instrument sensitivities can vary The injection time and voltage may be adjusted in the Module Manager A suggested range for the injection time is 3 22 seconds and for the injection voltage is 1 3kV 2 In the Protocol Manager select New Type a name for your protocol Select Regular in the Type drop down list and select the run module you created in the previous step in the Run Module drop down list Lastly select F in the Dye Set drop down list Select OK Page 12 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 prome
29. 0 1mM EDTA or nuclease free water Thermal cycler plate or tube problems Review the thermal cycling protocols in Section 4 B We have not tested other reaction tubes plates 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 16 HS Primer Pair for 15 seconds using a vortex mixer before use Poor capillary electrophoresis injection ILS 600 peaks also affected Re inject the sample Check the syringe for leakage Check the laser power Samples were not denatured completely Heat denature samples for the recommended time then cool on crushed ice or in an ice water bath immediately prior to loading the gel or capillary Poor quality formamide was used Use only Hi Di formamide when analyzing samples Page 35 Symptoms Causes and Comments Extra peaks visible in one or Contamination with another template DNA or previously all color channels amplified DNA Cross contamination can be a problem Use aerosol resistant pipette tips and change gloves regularly Samples were not denatured completely Heat denature samples for the recommended time and cool on crushed ice or in an ice water bath immediately prior to loading the gel or capillary Artifacts of STR amplification PCR amplification of STR systems sometimes generates artifacts that appear as faint peaks one repeat unit smaller than the allele Stutter band peak
30. 0 5 1 0ng in the desired template DNA volume Pipet 0 5 1 0ng of the diluted DNA into a reaction tube containing PCR amplification mix Note The 9947A DNA which is cell line derived will show allelic imbalance and imbalance between STR loci We supply the 9947A DNA as a positive control template to confirm that the correct STR profile is obtained not to show a balanced profile Do not use cell line derived DNA to determine sensitivity or verify balance Page 8 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 TMD022 Printed in USA 1 09 Table 1 PCR Amplification Mix for the PowerPlex 16 HS System PCR Amplification Mix Component1 Volume Per Reaction Water Amplification Grade to a final volume of 25 0 l PowerPlex HS 5X Master Mix 5 0 l PowerPlex 16 HS 10X Primer Pair Mix 2 5 l template DNA 0 5 1ng 2 3 up to 17 5 l total reaction volume 25 l 1Add Water Amplification Grade to the tube first then add PowerPlex HS 5X Master Mix and PowerPlex 16 HS 10X Primer Pair Mix The template DNA will be added at Step 6 2Store DNA templates in nuclease free water or TE 4 buffer 10mM Tris HCl pH 8 0 0 1mM EDTA If the DNA template is stored in TE buffer that is not pH 8 0 or contains a higher EDTA concentration the volume of DNA added should not exceed 20 of the final reacti
31. 2006 The DNA IQ System on the Tecan Freedom EVO 100 Profiles in DNA 9 1 8 10 41 Bjerke M et al 2006 Forensic application of the Maxwell 16 Instrument Profiles in DNA 9 1 3 5 42 Mandrekar P et al 2007 Introduction to Maxwell 16 low elution volume configuration for forensic casework Profiles in DNA 10 2 10 12 43 Gill P Jeffreys A J and Werrett D J 1985 Forensic application of DNA fingerprints Nature 318 577 9 44 Krenke B E et al 2005 Development of a novel fluorescent two primer approach to quantitative PCR Profiles in DNA 8 1 3 5 45 Krenke E et al 2008 Developmental validation of a real time PCR assay for the simultaneous quantification of total human and male DNA Forensic Sci Int Genet 3 14 21 Additional STR references can be found at www promega com geneticidentity 9 Appendix 9 A Preparing the PowerPlex 16 HS System PCR Amplification Mix A worksheet to calculate the required amount of each PCR amplification mix component is provided in Table 2 Multiply the volume l per reaction by the total number of reactions to obtain the final PCR amplification mix volume l 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 TMD022 1 09 Table 2 PCR Amplification Mix for the PowerPlex 16 HS System
32. 3 8 31 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 32 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 33 Sprecher C J et al 1996 General approach to analysis of polymorphic short tandem repeat loci BioTechniques 20 266 76 34 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 Page 43 35 Jones D A 1972 Blood samples Probability of discrimination J Forensic Sci Soc 12 355 9 36 Brenner C and Morris J W 1990 In Proceedings from the International Symposium on Human Identification 1989 Promega Corporation 21 53 37 Mandrekar P V Krenke B E and Tereba A 2001 DNA IQ The intelligent way to purify DNA Profiles in DNA 4 3 16 38 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 39 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 40 Cowan C
33. 4 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD022 1 09 Page 16 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 TMD022 Printed in USA 1 09 5 B Detection of Amplified Fragments Using the ABI PRISM 3100 Genetic Analyzer with Data Collection Software Version 1 0 1 or 1 1 continued 11 To collect 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 12 Select OK This new plate record will appear in the pending plate records table on the plate setup page of the collection software 13 Place samples in the instrument and close the instrument doors 14 Locate the pending plate record that you just created and click once on the name 15 Once the pending plate record is highlighted click on the plate graphic that corresponds to the plate on the autosampler that contains your amplified samples to link the plate to the plate record 16 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 17 Select Run Instrument on th
34. 7 1 in 2 93 1017 1 in 3 74 1017 Paternity Index 2 510 000 1 520 000 522 000 4 110 000 Power of Exclusion 0 9999996 0 9999994 0 9999983 0 9999998 Page 49 9 F Composition of Buffers and Solutions 9 G DNA Extraction and Quantitation Methods The DNA IQ System Cat DC6700 is a DNA isolation and quantitation system designed specifically for forensic and paternity samples 37 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 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 H 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
35. Master Mix PowerPlex 16 HS 10X Primer Pair Mix and 9947A DNA completely Note 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 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 consume a small amount of each reagent it ensures that you will have enough PCR amplification mix for all samples It also ensures that each reaction contains the same PCR amplification mix 3 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 GeneAmp PCR System 9600 or 9700 thermal cyclers use 0 2ml MicroAmp 8 strip reaction tubes or MicroAmp plate For the Perkin Elmer model 480 thermal cycler we recommend 0 5ml GeneAmp thin walled reaction tubes 4 Add the final volume of each reagent listed in Table 1 into a sterile 1 5ml amber colored tube Mix gently Table 1 shows the component volumes per reaction A worksheet to calculate the required amount of each PCR amplification mix component is provided in Section 9 A Table 2 Note In tests performed at Promega we have found that reactions can remain at room temperature for up t
36. PowerPlex16 advanced 6 Select the Allele tab Figure 3 7 Select the bin set corresponding to the PowerPlex System Promega_Bins_ID3 2 X where X refers to the most recent version of the bin set Page 20 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 TMD022 Printed in USA 1 09 5724TA Figure 3 The Allele tab Select the bin set Promega_Bins_ID3 2 X txt where X refers to the most recent version of the bin set Page 21 8 Ensure that the Use marker specific stutter ratio if available box is checked 9 Enter the values shown in Figure 3 for proper filtering of stutter peaks when using the PowerPlex 16 HS System For an explanation of the proper usage and effects of these settings refer to the Applied Biosystems user bulletin titled Installation Procedures and New Features for GeneMapper ID Software 3 2 Note Some of these settings have been optimized and are different from the recommended settings in the user bulletin 10 Select the Peak Detector tab We recommend the settings shown in Figure 4 Note Select full range or partial range for the analysis range When using a partial range choose an appropriate analysis range based on the data Choose a start point after the primer peak and just before the first defined internal lane standard pea
37. S 600 and 9 75 l of formamide 2 Mix for 10 15 seconds using a vortex mixer 3 Pipet 10 l of formamide internal lane standard mix into each well 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 TMD022 1 09 5 A Detection of Amplified Fragments Using the ABI PRISM 3100 or 3100 Avant Genetic Analyzer with Data Collection Software Version 2 0 and the Applied Biosystems 3130 or 3130xl Genetic Analyzer with Data Collection Software Version 3 0 continued 4 Add 1 l of amplified sample or 1 l of allelic ladder mix Cover wells with appropriate septa Note Instrument detection limits vary therefore injection time or the amount of product mixed with loading cocktail may need to be increased or decreased Use the Module Manager in the data collection software to modify the injection time or voltage in the run module If peak heights still are higher than desired use less DNA template in the amplification reactions or reduce the number of cycles in the amplification program by 2 4 cycles to achieve the desired signal intensity 5 Centrifuge plate briefly to remove air bubbles from the wells if necessary 6 Denature samples at 95 C for 3 minutes then immediately chill on crushed ice or in an ice water bath for 3 minutes Denature samples just prior to loading
38. SA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD022 Printed in USA 1 09 9 C Advantages of Using the Loci in the PowerPlex 16 HS System continued Table 4 The PowerPlex 16 HS System Allelic Ladder Information STR Locus Label Size Range of Allelic Ladder Components1 2 bases Repeat Numbers of Allelic Ladder Components3 Penta E FL 379 474 5 24 D18S51 FL 290 366 8 10 10 2 11 13 13 2 14 27 D21S11 FL 203 259 24 24 2 25 25 2 26 28 28 2 29 29 2 30 30 2 31 31 2 32 32 2 33 33 2 34 34 2 35 35 2 36 38 TH01 FL 156 195 4 9 9 3 10 11 13 3 D3S1358 FL 115 147 12 20 FGA TMR 322 444 16 18 18 2 19 19 2 20 20 2 21 21 2 22 22 2 23 23 2 24 24 2 25 25 2 26 30 31 2 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 Amelogenin4 TMR 106 112 X Y Penta D JOE 376 449 2 2 3 2 5 7 17 CSF1PO JOE 321 357 6 15 D16S539 JOE 264 304 5 8 15 D7S820 JOE 215 247 6 14 D13S317 JOE 176 208 7 15 D5S818 JOE 119 155 7 16 1The length of each allele in the allelic ladder has been confirmed by sequence analyses 2When 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
39. SA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD022 Printed in USA 1 09 9 Appendix 43 A Preparing the PowerPlex 16 HS System PCR Amplification Mix 43 B Advantages of STR Typing 44 C Advantages of Using the Loci in the PowerPlex 16 HS System 44 D Power of Discrimination 48 E The Internal Lane Standard 600 48 F Composition of Buffers and Solutions 49 G DNA Extraction and Quantitation Methods 49 H Related Products 50 1 Description STR 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 9
40. T e c h n i c a l M a n u a l PowerPlex 16 HS System INSTRUCTIONS FOR USE OF PRODUCTS DC2100 AND DC2101 PRINTED IN USA 1 09 Part TMD022 Page 1 1 Description 2 2 Product Components and Storage Conditions 4 3 Before You Begin 5 A Precautions 5 B Matrix Standardization or Spectral Calibration 6 4 Protocols for DNA Amplification Using the PowerPlex 16 HS System 6 A Amplification Setup 7 B Amplification Thermal Cycling 9 5 Instrument Setup and Sample Preparation 11 A Detection of Amplified Fragments Using the ABI PRISM 3100 or 3100 Avant Genetic Analyzer with Data Collection Software Version 2 0 and the Applied Biosystems 3130 or 3130xl Genetic Analyzer
41. allelic ladder with the base pair size and range listed in the categories for the same alleles If necessary increase the category start range in the category window to greater than 6bp and save the macro under a new name Allelic ladder peaks were too high causing stutter peaks to be called as allele peaks Use a shorter injection time decrease the amount of allelic ladder used or re analyze the allelic ladder sample using increased peak amplitude thresholds in the GeneScan analysis parameters Allelic ladder data were not compatible with the PowerTyper Macro file used Confirm that the PowerTyper Macro file matches the allelic ladder being used Page 41 Symptoms Causes and Comments The plots window or allele The macros were not run in the proper order Use the POWER table does not display all data or POWER 20 Filter macro option All four dye colors were not imported For Genotyper software versions 2 5 and 3 5 or higher set preferences in the Edit menu to import the blue green yellow and red colors The Check ILS macro All four dye colors were not imported For Genotyper displays an empty plot software versions 2 5 and 3 5 or higher set preferences in the window Edit menu to import the blue green yellow and red colors Off ladder peaks Migration of samples changed slightly over the course of a CE run with many samples This may be due to changes in temperature or the CE column over time Use a dif
42. ample DNA handling primer pairs assembling amplification reactions and analyzing amplification products Reagents and materials used prior to amplification PowerPlex HS 5X Master Mix PowerPlex 16 HS 10X Primer Pair Mix 9947A DNA and Water Amplification Grade are provided in a separate box and should be stored separately from those used following amplification PowerPlex 16 HS Allelic Ladder Mix and Internal Lane Standard 600 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 H Some reagents used in the analysis of STR products are potentially hazardous and should be handled accordingly Formamide is an irritant and a teratogen 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 formamide 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 TMD022 1 09 Page 6 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 TMD022 Printed in USA 1 09 3 B Matrix Standardization or Spectral Calibration
43. andard was not correctly defined or size peaks were missing Redefine size standard to include only peaks present in your sample Terminating analysis early or using short run times will cause larger ladder peaks to be missing This will cause your sizing quality to be flagged as red and no allele sizes will be called 5686TA Figure 12 An example showing improper assignment of size standard fragments in the GeneMapper ID software Symptoms Causes and Comments Error message The bin set assigned to the analysis method may have been Both the Bin Set used in the deleted In the GeneMapper Manager select the Analysis Analysis Method and the Panel Methods tab and open the analysis method of interest Select must belong to the same the Alleles tab and select an appropriate bin set Chemistry Kit The wrong bin set was chosen in the analysis method Allele tab Be sure to choose the appropriate bin set as shown in Figure 3 Significantly raised baseline Poor spectral calibration for the ABI PRISM 3100 and 3100 Avant Genetic Analyzers and Applied Biosystems 3130 and 3130xl Genetic Analyzers Perform a new spectral calibration and re run the samples Poor matrix for the ABI PRISM 310 Genetic Analyzer Re run and optimize the matrix Use of Classic mode analysis method Use of Classic mode analysis on samples can result in baselines with more noise than those analyzed using the Basic or Advanced mode analysis m
44. anel Manager icon in the upper left tile navigation pane 4 Select File then Import Panels 5 Navigate to the saved panel and bin files Select Promega_Panels_ID3 2 X txt where X refers to the most recent version of the panel and bin files Select Import 6 In the navigation pane highlight the Promega_Panels_ID3 2 X folder that you just imported 7 Select File then Import Bin Set 8 Navigate to the saved panel and bin files Select Promega_Bins_ID3 2 X txt then Import 9 At the bottom of the Panel Manager window select Apply then OK The panel manager window will close automatically 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 TMD022 1 09 6 B Creating a Casework Analysis Method with GeneMapper ID Software These instructions loosely follow the Applied Biosystems GeneMapper ID software tutorial pages 1 11 1 Select Tools then GeneMapper Manager 2 Select the Analysis Methods tab 3 Select New and a new analysis method dialog box will open 4 Select HID and select OK Note If you do not see the HID option you do not have the GeneMapper ID software Contact Applied Biosystems 5 Enter a descriptive name for the analysis method such as
45. ax 600 Size Calling Method Local Southern Method Split Peak Correction None 1Smooth options should be determined by individual laboratories Occasionally the TH01 alleles 9 3 and 10 will not be distinguished using heavy smoothing 2The peak amplitude thresholds are the minimum peak heights that the software will call as a peak Values for peak amplitude thresholds are usually 50 200RFU and should be determined by individual laboratories Page 29 6 F Sample Analysis Using the Genotyper Software and PowerTyper 16 Macro To facilitate analysis of data generated with the PowerPlex 16 HS System we have created a file to allow automatic assignment of genotypes using the Genotyper software After samples are amplified detected using the ABI PRISM 310 or 3100 Genetic Analyzer using data collection software version 1 0 1 or 1 1 and analyzed using the GeneScan analysis software sample files can be imported into the Genotyper program and analyzed using the PowerTyper 16 Macro Release 2 0 The PowerTyper 16 Macro Release 2 0 is available upon request from Promega The PowerTyper 16 Macro Release 2 0 is provided on the PowerTyper Macros CD ROM Cat DG3470 The PowerTyper Macros can be also downloaded from the Promega web site at www promega com geneticidtools The PowerTyper 16 Macro Release 2 0 is used in conjunction with Macintosh Genotyper software version 2 5 and Windows NT Ge
46. c that corresponds to the plate on the autosampler that contains your amplified samples 9 When the plate record is linked to the plate the plate graphic will change from yellow to green and the green Run Instrument arrow becomes enabled 10 Click on the green Run Instrument arrow on the toolbar to start the sample run 11 Monitor electrophoresis by observing the run view array or capillaries viewer window in the data collection software Each injection will take approximately 45 minutes 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 TMD022 1 09 Page 14 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 TMD022 Printed in USA 1 09 5 B Detection of Amplified Fragments Using the ABI PRISM 3100 Genetic Analyzer with Data Collection Software Version 1 0 1 or 1 1 Materials to Be Supplied by the User 95 C dry heating block water bath or thermal cycler crushed ice or ice water bath centrifuge compatible with 96 well plates aerosol resistant pipette tips 3100 capillary array 36cm performance optimized polymer 4 POP 4 for the 3100 10X genetic analyzer buffer with EDTA MicroAmp optical 96 well plate and septa
47. cation The saved file can be viewed and analyzed using Microsoft Excel 11 Save the analyzed data Go to the File menu and select Save as The PowerTyper Macro is a Genotyper file and can be overwritten if Save is used instead of Save as PowerTable Format Allele Table Format CODIS Table Format 6 G Controls 1 Observe the results for the negative control The negative control should be devoid of amplification products 2 Observe the results for the 9947A positive control DNA Compare the 9947A DNA allelic repeat sizes with the locus specific allelic ladder The expected 9947A DNA allele designations for each locus are listed in Table 5 Section 9 B The 9947A DNA which is cell line derived will show allelic imbalance and imbalance between 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 Printed in USA Part TMD022 1 09 Sample Info Sample Comment Category Peak 1 Peak 2 Peak 3 Peak 4 Over flow Low Signal Satura tion Edited Label Edited Row Sample Info Category Allele 1 Category Allele 2 Category Allele 1 Category Allele 2 Category Allele 1 Category Allele 2 Category Allele 1 Category Allele 2 Sample Info Category Peak 1 Peak 2 Page 32 Promega Corporation 2800 Woods Hollow Road
48. cause of the small amount of template used this is rarely a problem Depending on the DNA extraction procedure used and sample source inhibitors might be present in the DNA sample Insufficient template Use the recommended amount of template DNA Insufficient template Low copy number LCN analysis using capillary electrophoresis may benefit from reducing competing charged particles during injection This can be accomplished with post PCR cleanup or desalting lower conductivity formamide or reduced amounts of ILS 600 In house validation should be performed for any of these methods Insufficient enzyme activity Vortex the PowerPlex HS 5X Master Mix before use and use the recommended amount Incorrect amplification program Confirm the amplification program The PowerPlex HS 5X Master Mix was not vortexed well before use Vortex the 5X Master Mix for 5 10 seconds before dispensing into reaction tubes or plates An air bubble formed at the bottom of the reaction tube Use a pipette to remove the air bubble or centrifuge the reactions briefly before thermal cycling 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 Na Mg2 or EDTA from the DNA sample can negatively affect PCR A change in pH may also affect PCR Store DNA in TE 4 buffer 10mM Tris HCl pH 8 0
49. detection affecting the dye color to dye color balance 6 E Sample Analysis Using the GeneScan Software and Macintosh Operating Systems 1 Analyze data using the GeneScan analysis software 2 Review the raw data for one or more sample runs Highlight the sample file name then in 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 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 TMD022 1 09 Page 28 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 TMD022 Printed in USA 1 09 6 E Sample Analysis Using the GeneScan Software and Macintosh Operating Systems continued 3 The recommended analysis parameters are 4 The analysis parameters can be saved in the Params folder 5 Apply the stored analysis parameters file to the samples 6 Assign a new size standard Select a sample file highlight the arrow next to size standard then select define new Assign the size standard peaks as shown in Figur
50. ds 3100 3130 Cat DG4650 The quality of formamide is critical Use Hi Di formamide with a conductivity less than 100 S cm Freeze formamide in aliquots at 20 C Multiple freeze thaw cycles or long term storage at 4 C may cause a breakdown of the formamide Formamide with a conductivity greater than 100 S cm may contain ions that compete with DNA during injection which results in lower peak heights and reduced sensitivity A longer injection time may not increase the signal Caution Formamide is an irritant and a teratogen 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 formamide Sample Preparation 1 Prepare a loading cocktail by combining and mixing Internal Lane Standard 600 and Hi Di formamide as follows 0 5 l ILS 600 injections 9 5 l Hi Di formamide injections Note The volume of internal lane standard used in the loading cocktail can be increased or decreased to adjust the intensity of the size standard peaks The optimal peak height for the 100 base fragment of the internal lane standard is 500 1 000RFU If peak heights are too low we recommend altering the formamide internal lane standard mix to contain 1 0 l of ILS 600 and 9 0 l of Hi Di formamide If peak heights are too high we recommend altering the loading cocktail to contain 0 25 l of IL
51. e 13 in Section 9 E Store the size standard in the Size Standards folder 7 Apply the size standard file to the samples then analyze the sample files See Section 6 F for additional information on the use of the PowerTyper 16 Macro Release 2 0 and Genotyper software For additional information regarding the GeneScan analysis software refer to the GeneScan Analysis Software User s Manual Notes 1 Peak heights outside the linear range of the instrument may generate artifact peaks due to instrument saturation i e overloading the sample Bleedthrough pull ups from one color to another may be observed Saturated signal may also appear as two peaks split peak 2 If peak heights are not within the linear range of detection of the instrument the ratio of stutter peaks to real allele peaks increases and allele designations become difficult to interpret The balance of peak heights may also appear less uniform 3 There can be variation between instruments regarding the relative fluorescence levels detected using the same sample Furthermore different instruments vary in the relative efficiency of color detection affecting the dye color to dye color balance Analysis Range Start Defined in Step 2 Stop 10 000 Data Processing Baseline Checked Multicomponent Checked Smooth Options Light1 Peak Detection Peak Amplitude Thresholds2 B Y G R Min Peak Half Width 2pts Size Call Range Min 60 M
52. e toolbar to start the sample run 18 Monitor electrophoresis by observing the run status array and capillary views windows in the collection software Each injection will take approximately 45 minutes 5 C Detection of Amplified Fragments Using the ABI PRISM 310 Genetic Analyzer Materials to Be Supplied by the User 95 C dry heating block water bath or thermal cycler 310 capillaries 47cm 50 m performance optimized polymer 4 POP 4 10X genetic analyzer buffer with EDTA sample tubes and septa aerosol resistant pipette tips Hi Di formamide Applied Biosystems Cat 4311320 PowerPlex Matrix Standards 310 Cat DG4640 crushed ice or ice water bath The quality of formamide is critical Use Hi Di formamide with a conductivity less than 100 S cm Freeze formamide in aliquots at 20 C Multiple freeze thaw cycles or long term storage at 4 C may cause a breakdown of the formamide Formamide with a conductivity greater than 100 S cm may contain ions that compete with DNA during injection which results in lower peak heights and reduced sensitivity A longer injection time may not increase the signal Caution Formamide is an irritant and a teratogen 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 formamide Page 17 Sample Pr
53. elect OK Processing Sample Data for Casework 1 Import sample files into a new project as described in the Applied Biosystems GeneMapper ID Software Human Identification Analysis Tutorial 2 In the Sample Type column use the drop down menu to select Ladder Sample Positive Control or Negative Control Every folder in the project must contain at least one ladder that is designated as such for proper genotyping 3 In the Analysis Method column select the analysis method created previously in the Creating a Casework Analysis Method section 4 In the Panel column select PowerPlex_16_ID3 2 X where X refers to the most recent version of the panel files This is the panel set that was imported in Section 6 A 5 In the Size Standard column select the size standard that was created in the Creating a Size Standard section 6 If analyzing data from an ABI PRISM 310 Genetic Analyzer ensure that the appropriate matrix file is selected in the Matrix column 7 Select Analyze green arrow button to start data analysis Page 23 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 TMD022 1 09 5726TA Figure 6 The Size Standard Editor 5725TA Figure 5 The Select Dye and Analysis Method window Page 24 Promega Corporation
54. eparation 1 Prepare a loading cocktail by combining Internal Lane Standard 600 ILS 600 and Hi Di formamide as follows 1 0 l ILS 600 injections 24 0 l Hi Di formamide injections Note The volume of internal lane standard used in the loading cocktail can be increased or decreased to adjust the intensity of the size standard peaks The optimal peak height for the 100 base fragment of the internal lane standard is 500 1 000RFU If peak heights are too high we recommend altering the loading cocktail to contain 0 5 l of ILS 600 and 24 5 l of Hi Di formamide 2 Mix for 10 15 seconds using a vortex mixer 3 Combine 25 0 l of prepared loading cocktail and 1 0 l of amplified sample Note Instrument detection limits vary therefore injection time or the amount of product mixed with loading cocktail may need to be increased or decreased Use the Module Manager in the data collection software to modify the injection time or voltage in the run module If peak heights still are higher than desired use less DNA template in the amplification reactions or reduce the number of cycles in the amplification program by 2 4 cycles to achieve the desired signal intensity 4 Combine 25 0 l of prepared loading cocktail and 1 0 l of PowerPlex 16 HS Allelic Ladder Mix 5 Centrifuge tubes briefly to remove air bubbles from the wells if necessary 6 Denature samples and ladder by heating at 95
55. ethod Advanced mode analysis methods and size standards are recommended Red bar appears during analysis If none of the samples had matrices applied when run on the of samples and the following ABI PRISM 310 Genetic Analyzer no data will be displayed error message appears when data Apply a matrix file during analysis in the GeneMapper ID are displayed Some selected software and re analyze sample s do not contain analysis data Those sample s will not be shown Error message after attempting There was a conflict between different sets of panel and bin to import panel and bin files files Delete all panel and bin sets and re import files in a Unable to save panel data different order java SQLEException ORA 00001 unique constraint IFA CKP_NNN violated Allelic ladder peaks are GeneMapper ID software was not used or microsatellite labeled off ladder analysis settings were used instead of HID analysis settings GeneMapper software does not use the same algorithms as GeneMapper ID software and cannot correct for sizing differences using the allelic ladder Promega recommends using GeneMapper ID software to analyze PowerPlex reactions If using GeneMapper ID software version 3 2 be sure that the analysis method selected is an HID method This can be verified by opening the analysis method using the GeneMapper Manager then selecting the General tab The analysis type cannot be changed If the method
56. ferent injection of allelic ladder to determine sizes in the PowerTyper 16 Macro Release 2 0 Do not use the first injection on a new column for the ladder sample The base pair size of alleles was incorrect because incorrect fragment sizes were assigned to the internal lane standard Confirm that internal lane standard fragment sizes are assigned correctly Re analyze the sample using GeneScan software and redefine the internal lane standard fragments 8 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 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 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 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 6 Sambrook J Fritsch E F and Maniatis T 1989 Chapter 14 In vitro amplification of DNA by
57. from the same run The GeneMapper ID software requires that the allelic ladder be imported from the same folder as the sample Be sure that the allelic ladder is in the same folder as the sample Create a new project and re analyze as described in Section 6 B or 6 C Panel file selected for analysis was incorrect for the STR system used Assign correct panel file that corresponds to the STR system used for amplification The allelic ladder was not identified as an allelic ladder in the sample type column The wrong analysis type was chosen for the analysis method Be sure to use the HID analysis type The internal lane standard was not properly identified in the sample Manually redefine the sizes of the size standard fragments in the sample 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 TMD022 1 09 5685TA Figure 11 The error message that appears in the GeneMapper ID software when the analysis parameters and size standard have different analysis types Page 38 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 TMD022 Printed in USA 1 09 7 B GeneMapper ID Analysis Software continued Symptoms Causes and Comments Size standard not called Starting data
58. ga com Part TMD022 Printed in USA 1 09 Page 13 3 In the Plate Manager create a new plate record as described in the instrument user s manual In the dialog box that appears select GeneMapper Generic in the Application drop down list and select the appropriate plate type 96 well Add entries in the owner and operator windows and select OK Note If autoanalysis of sample data is desired refer to the instrument user s manual for instructions 4 In the GeneMapper plate record enter sample names in the appropriate cells Scroll to the right In the Results group 1 column select the desired results group In the Instrument Protocol 1 column select the protocol you created in Step 2 Be sure this information is present for each row that contains a sample name Select OK Note To create a new results group select New in the drop down menu in the results group column Select the General tab and enter a name Select the Analysis tab and select GeneMapper Generic in the Analysis type drop down list 5 Place samples in the instrument and close the instrument doors 6 In the spectral viewer confirm that dye set F is active and set the correct active calibration for dye set F 7 In the run scheduler locate the plate record that you just created in Steps 3 and 4 and click once on the name to highlight it 8 Once the plate record is highlighted click the plate graphi
59. he Matrix column 7 Select Analyze green arrow button to start the data analysis 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 TMD022 1 09 5785TA Figure 7 The Allele tab with settings for using a 20 peak filter Select the bin set Promega_Bins_ID3 2 X txt where X refers to the most recent version of the bin set Page 26 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 TMD022 Printed in USA 1 09 6 D Sample Analysis Using the GeneScan Software and Windows Operating Systems 1 Analyze data using the GeneScan analysis software 2 Review the raw data for one or more sample runs Highlight the sample file name then in 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 3 The recommended analysis parameters are shown in Figure 8 5684TA Figure 8 The Analysis Parameters window The start point of the analysis range which will vary is defined in Section 6 D Step 2 Page 27 4
60. in USA 1 09 8 References continued 14 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 15 Nakamura Y et al 1987 Variable number of tandem repeat VNTR markers for human gene mapping Science 235 1616 22 16 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 17 Levinson G and Gutman G A 1987 Slipped strand mispairing A major mechanism for DNA sequence evolution Mol Biol Evol 4 203 21 18 Schlotterer C and Tautz D 1992 Slippage synthesis of simple sequence DNA Nucleic Acids Res 20 211 5 19 Smith J R et al 1995 Approach to genotyping errors caused by nontemplated nucleotide addition by Taq DNA polymerase Genome Res 5 312 7 20 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 21 Walsh P S Fildes N J and Reynolds R 1996 Sequence analysis and characterization of stutter products at the tetranucleotide repeat locus vWA Nucleic Acids Res 24 2807 12 22 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 23 Brinkmann
61. k to help ensure proper sizing of the internal lane standard 11 Select the Peak Quality tab You may change the settings for peak quality Note For Steps 11 and 12 see the GeneMapper ID user s manual for more information 12 Select the Quality Flags tab You may also change these settings 13 Select OK to save your settings 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 TMD022 1 09 5723TA Figure 4 The Peak Detector tab Page 22 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 TMD022 Printed in USA 1 09 6 B Creating a Casework Analysis Method with GeneMapper ID Software continued Creating a Size Standard 1 Select Tools then GeneMapper Manager 2 Select the Size Standard tab 3 Select New 4 Select Basic or Advanced Figure 5 The type of analysis method selected must match the type of analysis method created earlier Select OK 5 Enter a detailed name such as ILS 600 advanced in the Size Standard Editor Figure 6 6 Choose red as the color for the size standard dye 7 Enter the sizes of the internal lane standard fragments see Section 9 E Figure 13 8 S
62. l lane standard i e ILS 600 in the red dye color Scroll down to view and confirm that the internal lane standard fragment sizes are correct If necessary re analyze samples using the GeneScan software and redefine internal lane standard fragments Note The software uses one ladder sample to determine allele sizes The macro uses the first ladder sample imported for allele designations 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 TMD022 1 09 Page 30 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 TMD022 Printed in USA 1 09 6 F Sample Analysis Using the Genotyper Software and PowerTyper 16 Macro continued 5 For casework double click on the POWER macro The POWER macro identifies alleles in the ladder sample and calculates offsets for all loci This process may take several minutes When completed a plots window will open to display the allelic ladders i e Penta E D18S51 D21S11 TH01 and D3S1358 Alternatively for databasing or paternity double click on the POWER 20 Filter macro This macro has a higher level of filtering than the standard POWER macro to reduce the need for manual editing of peak labels The POWER 20 Filter should no
63. le files containing allelic ladder All four dye colors were not imported For Genotyper software versions 2 5 and 3 5 or higher set preferences in the Edit menu to import the blue green yellow and red colors Error message Peak heights for one or more alleles in the allelic ladder Could not complete the sample file were below 150RFU The allelic ladder categories Run Macro command are defined as having a minimum peak height of 150RFU If because the labeled peak peak heights of ladder alleles are below 150RFU the software could not be found will not be able to locate the allele peak Re run the allelic ladder using more sample or longer injection time to assure peak heights above 150RFU CE spikes in the allelic ladder sample were identified as alleles by the macro Use a different injection of allelic ladder TH01 9 3 and 10 alleles were not separated when using heavy smoothing in the GeneScan analysis parameters Use light smoothing in the GeneScan analysis parameters Allelic ladder data were not compatible with the PowerTyper file used Confirm that the PowerTyper Macro file matches the allelic ladder being used The base pair size of alleles in the allelic ladder are outside of the defined category range Be sure internal lane standard fragments are correctly sized Redefine internal lane standard fragments and re analyze the sample using GeneScan software Compare the size of the smallest allele in the
64. llary was of poor quality or denaturation was ineffective See Section 7 for more information on how to minimize these artifacts 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 TMD022 1 09 5682TA A B C Figure 10 The PowerPlex 16 HS Allelic Ladder Mix The PowerPlex 16 HS Allelic Ladder Mix was analyzed with an Applied Biosystems 3130 Genetic Analyzer using a 3kV 5 second injection The sample file was analyzed with the GeneMapper ID software version 3 2 and PowerPlex 16 panel and bin files Panel A The fluorescein labeled allelic ladder components and their allele designations Panel B The JOE labeled allelic ladder components and their allele designations Panel C The TMR labeled allelic ladder components and their allele designations Page 34 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 TMD022 Printed in USA 1 09 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 7 A Amplification and Fragment Detection Symptoms Causes and Comments Faint or absent allele peaks Impure template DNA Be
65. loci to evaluate DNA mixtures often encountered in forensic casework Finally the Amelogenin locus is included in the PowerPlex 16 HS System to allow gender identification of each sample Table 5 lists the PowerPlex 16 HS System alleles revealed in commonly available standard DNA templates We have carefully selected STR loci and primers to avoid or minimize artifacts including those associated with Taq DNA polymerase such as repeat slippage and terminal nucleotide addition Repeat slippage 17 18 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 the DNA sequence being amplified Page 45 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 TMD022 1 09 Table 3 The PowerPlex 16 HS System Locus Specific Information STR Locus Label Chromosomal Location GenBank Locus and Locus Definition Repeat Sequence1 5 3 Penta E FL 15q NA AAAGA D18S51 FL 18q21 3 HUMUT574 AGAA 24 D21S11 FL 21q11 21q21 HUMD21LOC TCTA Complex 24 TH01 FL 11p15 5 HUMTH01 human tyrosine hydroxylase gene AATG 24 D3S1358 FL 3p NA TCTA Complex FGA TMR 4q28 HUMFIBRA human
66. ltModule module run time to 1 800 seconds 3 Change the injection voltage to 3kV 4 Change the injection time to 11 seconds Note Instrument sensitivities can vary Injection time and voltage may be adjusted in the Module Manager A suggested range for the injection time is 3 22 seconds and for the injection voltage is 1 3kV 5 Save the module with a new name e g GeneScan36_POP4PowerPlex16_3kV_11secs_1800 Use this as the initial run module for all runs 6 Open a new plate record Name the plate and select GeneScan Select the plate size 96 well Select Finish 7 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 entered to successfully analyze data with the PowerTyper 16 Macro Release 2 0 8 In the BioLIMS Project column select 3100_Project1 from the pull down menu 9 In the Dye Set column select Z from the pull down menu 10 When using the ABI PRISM 3100 data collection software version 1 0 1 or 1 1 select GeneScan36_POP4PowerPlex16_3kV_11secs_1800 from the pull down menu in the Run Module 1 column Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 27
67. m 9600 and 9700 thermal cyclers For information on other thermal cyclers please contact Promega Technical Services by e mail genetic promega com Amplification and detection instrumentation may vary You may need to optimize protocols including cycle number and injection time or loading volume for each laboratory instrument Testing at Promega Corporation shows that 10 22 cycles work well for 0 5ng of purified DNA templates For higher amounts of input DNA i e FTA paper or to decrease sensitivity fewer cycles such as 10 16 10 18 or 10 20 should be evaluated In house validation should be performed 1 Place the tubes or MicroAmp plate in the thermal cycler 2 Select and run a recommended protocol The preferred protocols for use with the GeneAmp PCR System 9600 and 9700 thermal cyclers and Perkin Elmer model 480 thermal cycler are provided below 3 After completion of the thermal cycling protocol store the amplified 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 TMD022 1 09 Page 10 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 pr
68. mamide If peak heights are too high we recommend altering the loading cocktail to contain 0 25 l of ILS 600 and 9 75 l of formamide 2 Mix for 10 15 seconds using a vortex mixer 3 Pipet 10 l of formamide internal lane standard mix into each well Page 15 4 Add 1 l of amplified sample or 1 l of allelic ladder mix Cover wells with appropriate septa Note Instrument detection limits vary therefore injection time or the amount of product mixed with loading cocktail may need to be increased or decreased Use the Module Manager in the data collection software to modify the injection time or voltage in the run module If peak heights are higher than desired use less DNA template in the amplification reactions or reduce the number of cycles in the amplification program by 2 4 cycles to achieve the desired signal intensity 5 Centrifuge plate briefly to remove air bubbles from the wells if necessary 6 Denature samples at 95 C for 3 minutes then immediately chill on crushed ice or in an ice water bath for 3 minutes Denature samples just prior to loading the instrument Instrument Preparation Refer to the ABI PRISM 3100 Genetic Analyzer User s Manual for instructions on cleaning the blocks installing the capillary array performing a spatial calibration and adding polymer to the reserve syringe 1 Open the ABI PRISM 3100 data collection software 2 Change the GeneScan36_POP4Defau
69. mine sizes Poor injection of allelic ladder Include more than one ladder per instrument run Peak height imbalance Excessive amount of DNA Amplification of gt 1ng 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 by 2 4 cycles 10 20 or 10 18 cycling to improve locus to locus balance Note Dilution of overamplified samples can result in dropout of larger loci Use of FTA paper Results may be similar to those obtained with excess amounts of DNA template Reduce the number of cycles in the amplification program by 2 4 cycles 10 20 or 10 18 cycling to improve locus to locus balance Degraded DNA sample DNA template is degraded and larger loci show diminished yield Repurify the template DNA 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 5X Master Mix completely and vortex for 15 seconds before use 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 instances Impure DNA template Inhibitors that may be present in forensic samples can lead to allele dropout or imbalance Impure DNA template Include a protei
70. mize the injection or gel loading conditions See Section 5 CE related artifacts contaminants Contaminants in the water used with the instrument or to dilute the 10X genetic analyzer buffer may generate peaks in the blue and green dye colors Use autoclaved water change vials and wash buffer reservoir Long term storage of amplified sample in formamide can result in degradation Repeat sample preparation using fresh formamide The CE polymer was beyond its expiration date or polymer was stored at room temperature for more than one week Maintain instrumentation on a daily or weekly basis as recommended by the manufacturer 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 TMD022 1 09 7 A Amplification and Fragment Detection continued Symptoms Causes and Comments Allelic ladder not running Allelic ladder and primer pair mix were not compatible Ensure the same as samples that the allelic ladder is from the same kit as the primer pair mix Poor quality formamide Use only Hi Di formamide when analyzing samples Be sure the allelic ladder and samples are from the same instrument run Migration of samples changed slightly over the course of a CE run with many samples This may be due to changes in temperature or the CE column over time Use a different injection of allelic ladder to deter
71. mp or 0 2ml MicroAmp reaction tubes or MicroAmp optical 96 well reaction plate Applied Biosystems 1 5ml amber colored microcentrifuge tubes Fisher Cat 05 402 26 aerosol resistant pipette tips see Section 9 H Mineral Oil Cat DY1151 for use with the model 480 thermal cycler We routinely amplify 0 5ng of template DNA in a 25 l reaction volume using the protocols detailed below With gt 1ng of DNA preferential amplification of smaller loci may occur Expect to see high peak heights at the smaller loci and relatively lower peak heights at the 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 The PowerPlex 16 HS System is optimized for the GeneAmp PCR System 9700 thermal cycler Amplification protocols for the GeneAmp PCR Systems 9600 thermal cycler and Perkin Elmer model 480 thermal cycler are provided Page 7 4 A Amplification Setup The use of gloves and aerosol resistant pipette tips is highly recommended to prevent cross contamination Keep all pre amplification and post amplification reagents in separate rooms Prepare amplification reactions in a room dedicated for reaction setup 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 A 1 Thaw the PowerPlex HS 5X
72. nase K digestion prior to DNA purification PCR amplification mix prepared in Section 4 A was not mixed well Vortex the PCR amplification mix for 5 10 seconds before dispensing into the reaction tubes or plate Page 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 Part TMD022 Printed in USA 1 09 Page 37 7 B GeneMapper ID Analysis Software Symptoms Causes and Comments Alleles not called To analyze samples with GeneMapper ID software the analysis parameters and size standard must both have Basic or Advanced as the analysis type If they are different an error is obtained Figure 11 To analyze samples with GeneMapper ID software at least one allelic ladder must be defined An insufficient number of ILS 600 fragments was defined Be sure to define at least one ILS 600 fragment smaller than the smallest sample peak and at least one ILS 600 fragment larger than the largest sample peak Run was too short and larger peaks in ILS were not captured Not all ILS 600 peaks defined in the size standard were detected during the run Create a new size standard using the internal lane standard fragments present in the sample Re run samples using a longer run time Off ladder alleles An allelic ladder from a different run than the samples was used Re analyze samples with an allelic ladder
73. notyper software version 3 6 or later The Genotyper software must be installed on your computer before the PowerTyper 16 Macro Release 2 0 can be used Be certain the sample info Macintosh computers or color info Windows NT operating systems column for each lane containing allelic ladder mix contains the word ladder The macro uses the word ladder to identify the sample file s containing allelic ladder Sample info can be added or modified after importing into the PowerTyper Macro Highlight the sample then select show dye lanes window in the Views menu 1 Transfer the PowerTyper 16 Macro Release 2 0 from the PowerTyper Macros CD ROM Cat DG3470 to a designated location on your computer hard drive Alternatively download the PowerTyper 16 Macro Release 2 0 from the Promega web site 2 Open the Genotyper software then the PowerTyper 16 Macro Release 2 0 For questions about the Genotyper software refer to the Genotyper Analysis Software User s Manual 3 In the File menu select Import and import the GeneScan project or sample files to be analyzed Import the blue yellow green and red dye colors Note To select the dye colors to be imported select Set Preferences in the Edit menu 4 Double click on the 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 interna
74. o 8 hours after reaction assembly and prior to thermal cycling with no adverse effect on amplification results Amplification of gt 1 0ng of DNA template may result in an imbalance in peak heights from locus to locus The smaller loci show greater amplification yield than the larger loci Reducing the number of cycles in the amplification program by 2 to 4 cycles i e 10 20 or 10 18 cycling can improve locus to locus balance 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 TMD022 1 09 4 A Amplification Setup continued 5 Vortex the PCR amplification mix for 5 10 seconds then pipet PCR amplification mix into each reaction tube Failure to vortex the PCR amplification mix sufficiently can result in poor amplification peak height imbalance and extra peaks 6 Pipet the template DNA 0 5ng for each sample into the respective tube containing PCR amplification mix Note Apparent DNA concentrations can differ depending on the DNA quantification method used 13 The amount of DNA template recommended here is based on DNA concentrations determined by measuring absorbance at 260nm We strongly recommend that you perform experiments to determine the optimal DNA amount for your particular DNA quantification method 7 For the positive amplification control dilute 9947A DNA to
75. o enrich for sperm cells in the presence of an excess of epithelial cells 43 Traditionally these samples are processed by performing a controlled lysis of epithelial cells in the absence of a reducing agent and centrifuging the samples to separate the pellet of intact sperm and cell debris from the buffer containing the DNA from lysed epithelial cells However this method is time consuming and labor intensive and several washings and recentrifugations often are required to obtain sperm free of epithelial DNA The Differex System simplifies differential extraction This system uses a standard proteinase K digestion and a combination of phase separation and differential centrifugation to separate sperm and epithelial DNA quickly and easily The Differex System offers similar recovery as the standard method commonly used for differential extraction The Differex System in combination with the DNA IQ System can be automated to extract up to 48 differential extractions in less than 5 hours including incubation time and less than 1 hour of hands on laboratory time For applications requiring human specific DNA quantification the Plexor HY System Cat DC1000 has been developed 44 45 See Section 9 H for ordering information 9 H Related Products Fluorescent STR Multiplex Systems Product Size Cat PowerPlex 16 Monoplex System Penta E Fluorescein 100 reactions DC6591 PowerPlex 16 Monoplex System Penta D JOE
76. omega com Part TMD022 Printed in USA 1 09 Protocol for the GeneAmp PCR System 9600 Thermal Cycler Protocol for the GeneAmp PCR System 9700 Thermal Cycler1 96 C for 2 minutes 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 22 cycles then 60 C for 30 minutes 4 C soak 96 C for 2 minutes 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 22 cycles then 60 C for 30 minutes 4 C soak Protocol for the Perkin Elmer Model 480 Thermal Cycler 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 22 cycles then 60 C for 30 minutes 4 C soak 1When using the GeneAmp PCR System 9700 thermal cycler the ramp rates indicated in the cycling program must be set and the 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
77. on 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 the extraction procedure used 3Apparent DNA concentrations can differ depending on the DNA quantification method used The amount of DNA template recommended here is based on DNA concentrations determined by measuring absorbance at 260nm We strongly recommend that you perform experiments to determine the optimal DNA amount for your particular DNA quantification method Page 9 8 For the negative amplification control pipet Water Amplification Grade or TE 4 buffer instead of template DNA into a reaction tube containing PCR amplification mix 9 If using the GeneAmp PCR System 9600 or 9700 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 tube and form an overlay to limit sample loss or cross contamination due to splattering 4 B Amplification Thermal Cycling This manual contains protocols for use of the PowerPlex 16 HS System with the Perkin Elmer model 480 and GeneAmp PCR syste
78. ons yet unknown the highly polymorphic Penta E locus does not display frequent microvariants Table 4 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 TMD022 1 09 Table 5 The PowerPlex 16 HS System Allele Determinations in Commonly Available Standard DNA Templates STR Locus Standard DNA Templates1 K5622 9947A 99483 Penta E 5 14 12 13 11 11 D18S51 15 16 15 19 15 18 D21S11 29 30 31 30 30 29 30 TH01 9 3 9 3 8 9 3 6 9 3 D3S1358 16 16 14 15 15 17 FGA 21 24 23 24 24 26 TPOX 8 9 8 8 8 9 D8S1179 12 12 13 13 12 13 vWA 16 16 17 18 17 17 Amelogenin X X X X X Y Penta D 9 13 12 12 8 12 CSF1PO 9 10 10 12 10 11 12 D16S539 11 12 11 12 11 11 D7S820 9 11 10 11 11 11 D13S317 8 8 11 11 11 11 D5S818 11 12 11 11 11 13 1Information 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 Information about the use of 9947A and 9948 DNA as standard DNA templates can be found in reference 27 2Strain K562 displays three alleles at the D21S11 locus 3Strain 9948 displays three alleles at the CSF1PO locus The peak height for allele 12 is much lower than tho
79. plified Fragments Using the ABI PRISM 310 Genetic Analyzer continued 3 Create a new GeneScan injection list Select the appropriate sample sheet by using the pull down menu 4 Select the GS STR POP4 1ml A Module using the pull down menu Change the injection time to 3 seconds and the run time to 30 minutes Keep the settings for the remaining parameters as shown below Inj Secs 3 Inj kV 15 0 Run kV 15 0 Run C 60 Run Time 30 You may need to optimize the injection time for individual instruments Injection times of 2 5 seconds are suggested for samples that contain 0 5 1ng of template DNA Note Migration of fragments may vary slightly over the course of a long ABI PRISM 310 Genetic Analyzer run This may be due to changes in temperature or changes in the column When analyzing many samples injections of allelic ladder at different times throughout the run can aid in accurately genotyping samples 5 Select the appropriate matrix file Section 3 B 6 To analyze data automatically select the auto analyze checkbox and the appropriate analysis parameters and size standard Refer to the ABI PRISM 310 Genetic Analyzer User s Manual for specific information on these options 7 After loading the sample tray and closing the doors select Run to start the capillary electrophoresis system 8 Monitor electrophoresis by observing the raw data and status windows Each sample will take ap
80. point was incorrect for the partial range chosen correctly Figure 12 in Section 6 B Adjust the starting data point in the analysis method Alternatively use a full range for the analysis Extra peaks in advanced mode size standard Open the size match editor Highlight the extra peak select Edit and select delete size label Select auto adjust sizes Run was too short and larger peaks in ILS were not captured Not all ILS 600 peaks defined in the size standard were detected during the run Create a new size standard using the internal lane standard fragments present in the sample Re run samples using a longer run time Peaks in size standard missing If peaks are below threshold decrease the peak threshold in the analysis method for the red channel to include peaks If peaks are low quality redefine the size standard for the sample to skip these peaks Error message The size standard and analysis method were not in the same Either panel size standard mode Classic vs Basic or Advanced Be sure both files or analysis method is invalid are set to the same mode either Classic or Basic or Advanced mode No alleles called but no error Panel was not selected for sample In the Panel column select message appears the appropriate panel set for the STR system that was used No size standard was selected In the size standards column be sure to select the appropriate size standard Size st
81. proximately 40 minutes for syringe pumping sample injection and sample electrophoresis Page 19 6 Data Analysis 6 A PowerPlex Panel and Bin Sets with GeneMapper ID Version 3 2 To facilitate analysis of data generated with the PowerPlex 16 HS System we have created panel and bin files to allow automatic assignment of genotypes using GeneMapper ID software version 3 2 We recommend that users of GeneMapper ID software version 3 2 complete the Applied Biosystems GeneMapper ID Software Human Identification Analysis Tutorial to familiarize themselves with proper operation of the software For GeneMapper ID software version 3 1 users we recommend upgrading to version 3 2 Getting Started 1 Obtain the proper panel and bin files for use with GeneMapper ID from the Promega web site at www promega com geneticidtools panels_bins 2 Enter your contact information and select GeneMapper ID version 3 2 Select Submit 3 Select the PowerPlex Panels amp Bin Sets link and save the zip file to your computer 4 Open the files using the Windows WinZip program and save the unzipped files to a known location on your computer Importing Panel and Bin Files These instructions loosely follow the Applied Biosystems GeneMapper ID software tutorial pages 1 4 1 Open the GeneMapper ID software version 3 2 2 Select Tools then Panel Manager 3 Highlight the P
82. ps pack Cat ART 10 Ultramicro Pipet Tip 0 5 10 l 960 DY1051 ART 20E Ultramicro Pipet Tip 0 5 10 l 960 DY1061 ART 20P Pipet Tip 20 l 960 DY1071 ART GEL Gel Loading Pipet Tip 100 l 960 DY1081 ART 100 Pipet Tip 100 l 960 DY1101 ART 100E Pipet Tip 100 l 960 DY1111 ART 200 Pipet Tip 200 l 960 DY1121 ART 1000E Pipet Tip 1 000 l 800 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 Part TMD022 1 09 Page 52 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 TMD022 Printed in USA 1 09 a 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 covering the foundational PCR process expired on March 29 2005 b U S Pat Nos 6 238 863 and 6 767 703 Korean Pat No
83. se for alleles 10 and 11 Page 48 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 TMD022 Printed in USA 1 09 9 D Power of Discrimination The fifteen STR loci amplified with the PowerPlex 16 HS System provide powerful discrimination Population statistics for these loci and their various multiplex combinations are displayed in Table 6 These data were generated as part of a collaboration 28 with The Bode Technology Group Springfield VA North Carolina Bureau of Investigation Raleigh NC Palm Beach County Sheriff s Office West Palm Beach FL Virginia Division of Forensic Science Richmond VA and Charlotte Mecklenburg Police Department Laboratory NC Data generation included analysis of over 200 individuals from African American Caucasian American and Hispanic American populations Data for Asian Americans include analysis of more than 150 individuals For additional population data for STR loci see references 29 34 and the Short Tandem Repeat DNA Internet DataBase at www cstl nist gov div831 strbase Table 6 shows the matching probability 35 for the PowerPlex 16 HS System in various populations The matching probability of the PowerPlex 16 HS System ranges from 1 in 1 83 1017 for Caucasian Americans to 1 in 1 41 1018 for African Americans A measure of discrimination often used
84. stered trademark of Apple Computer Inc Microsoft Windows and Windows NT are registered trademarks of Microsoft Corporation Products may be covered 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
85. t be used if mixtures may exist In general allelic ladders contain fragments of the same lengths as many known alleles for the locus Allelic ladder sizes and repeat units are listed in Table 4 Section 9 B Analysis using GeneScan analysis software and Genotyper software allows allele determination by comparing amplified sample fragments with allelic ladders and internal lane standards When using an internal lane standard the calculated lengths of allelic ladder components might differ from those listed in the table This is due to differences in migration resulting from sequence differences between the allelic ladder fragments and internal size standard and is not a matter of concern 6 Double click on the Allelic Ladders macro A plots window will open to display the blue fluorescein dye allelic ladders i e Penta E D18S51 D21S11 TH01 and D3S1358 green JOE dye allelic ladders i e Penta E CSF1PO D16S539 D7S820 D13S317 and D5S818 and yellow TMR dye allelic ladders i e FGA TPOX D8S1179 vWA and Amelogenin Confirm that the correct allele designations were assigned to the allelic ladders Figure 10 in Section 6 H Note The software uses one ladder sample to determine allele sizes The macro uses the first ladder sample imported for allele designations If the POWER macro is run a second time the software will use the second ladder if the POWER macro is run a third time the software will use the third ladder
86. the polymerase chain reaction In Molecular Cloning A Laboratory Manual Second Edition Cold Spring Harbor Laboratory Press Cold Spring Harbor New York 7 PCR Technology Principles and Applications for DNA Amplification 1989 Erlich H A ed Stockton 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 Butler J M 2005 Forensic DNA Typing 2nd ed Elsevier Academic Press London 10 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 11 Hartmann J M et al 1991 Guidelines for a quality assurance program for DNA analysis Crime Laboratory Digest 18 44 75 12 Internal Validation of STR Systems Reference Manual GE053 Promega Corporation 13 Kline M C et al 2005 Results from the NIST 2004 DNA quantitation study J Forensic Sci 50 571 8 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 TMD022 1 09 Page 42 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 TMD022 Printed
87. with Data Collection Software Version 3 0 11 B Detection of Amplified Fragments Using the ABI PRISM 3100 Genetic Analyzer with Data Collection Software Version 1 0 1 or 1 1 14 C Detection of Amplified Fragments Using the ABI PRISM 310 Genetic Analyzer 16 6 Data Analysis 19 A PowerPlex Panel and Bin Sets with GeneMapper ID Version 3 2 19 B Creating a Casework Analysis Method with GeneMapper ID Software 20 C Creating a Databasing or Paternity Analysis Method with GeneMapper ID Software 24 D Sample Analysis Using the GeneScan Software and Windows Operating Systems 26 E Sample Analysis Using the GeneScan Software and Macintosh Operating Systems 27 F Sample Analysis Using the Genotyper Software and PowerTyper 16 Macro 29 G Controls

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