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PowerPlex® Y23 System

1. 59 7 F GeneMapper ID Software 61 8 References 64 9 Appendix 66 9 A Advantages of Using the Loci in the PowerPlex Y23 System 66 9 B Detection of Ampli ed Fragments Using the Applied Biosystems 3130 or 3130xl Genetic Analyzer with POP 7 Polymer and Data Collection Software Version 3 0 70 9 C DNA Extraction and Quanti cation Methods and Automation Support 73 9 D The CC5 Internal Lane Standard 500 Y23 74 9 E Composition of Bu ers and Solutions 74 9 F Related Products 75 9 G Summary of Changes 76 Promega Corpora on
2. nished the Analysis Summary screen will appear We recommend that you review any yellow or red marker header bars in the plots view and handle them according to laboratory standard operating procedures The values displayed in the Analysis Method Peak Quality and SQ amp GQ Settings tabs are defaults and will a ect the quality values displayed in the plot settings We recommend that you modify the values in these tabs to t your laboratory s data analysis protocols 6 E Creating a Databasing or Paternity Analysis Method Using a Global Filter with GeneMapper ID X Software Version 1 2 These instructions are intended as a guide to start analyzing data in GeneMapper ID X software They are not intended as a comprehensive guide for using the GeneMapper ID X software We recommend that users contact Applied Biosystems for training on the software 1 Select Tools then GeneMapper ID X Manager 2 Select the Analysis Methods tab 3 Select New and a new analysis method dialog box will open 4 In the Analysis Method Editor window select GeneMapper ID X Security Group as the Security Group This allows access for all users of the software Other security groups may be used 5 Enter a descriptive name for the analysis method such as PowerPlexY23 20 Filter 6 Select the Allele tab Figure 16 7 Select the bins text le that was imported in Section 6 A 8 Ensure that the U
3. 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 3 www promega com TMD035 Revised 3 15 The PowerPlex Y23 System provides all materials necessary to amplify Y STR regions of human genomic DNA including a hot start DNA polymerase which is a component of the PowerPlex Y23 5X Master Mix This manual contains protocols for use of the PowerPlex Y23 System with the GeneAmp PCR System 9700 thermal cycler in addition to protocols to separate ampli ed products and detect separated material Figure 1 Protocols to operate the uorescence detection instruments should be obtained from the instrument manufacturer Information about other Promega uorescent STR systems is available upon request from Promega or online at www promega com products genetic identity Ampli cation Setup Instrument Setup and Sample Preparation Thermal Cycling Section 4 Section 4 Sections 5 and 9 GeneAmp PCR System 9700 Data Analysis Section 6 Applied Biosystems 3500 or 3500xL Genetic Analyzer with 3500 Data Collection Software Version 1 0 Section 5 A GeneMapper ID X Software Version 1 2 Applied Biosystems 3130 or 3130xl Genetic Analyzer with POP 4 Polymer and Data Collection Software Version 3 0 Section 5 B Applied Biosystems 3130 or 3130xl Genetic Analyzer with POP 7 Polymer and Data Collection Software Version 3 0 Section 9 B
4. 4 25 l PowerPlex Y23 Allelic Ladder Mix 2 300 l CC5 Internal Lane Standard 500 Y23 The PowerPlex Y23 Allelic Ladder Mix is provided in a separate sealed bag for shipping This component should be moved to the post ampli cation box after opening The Water Ampli cation Grade is provided in a separate sealed bag for shipping This component should be moved to the pre ampli cation box after opening Storage Conditions For long term storage store all components except the 2800M Control DNA at 30 C to 10 C in a nonfrost free freezer Store the 2800M Control DNA at 2 10 C For daily use the PowerPlex Y23 System components can be stored for up to 1 month at 2 10 C The PowerPlex Y23 10X Primer Pair Mix PowerPlex Y23 Allelic Ladder Mix and CC5 Internal Lane Standard 500 Y23 CC5 ILS 500 Y23 are light sensitive and must be stored in the dark We strongly recommend that pre ampli cation and post ampli cation reagents be stored and used separately with di erent pipettes tube racks etc Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 5 www promega com TMD035 Revised 3 15 Available Separately The proper panels bins and stutter text les and size standard xml le for use with GeneMapper ID and ID X software can be downloaded at www promega com resources tools genemapper id software
5. Done to save changes and close the GeneMapper ID X Manager 30 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD035 Revised 3 15 www promega com 6 C Creating a Size Standard with GeneMapper ID X Software Version 1 2 1 Select Tools then GeneMapper ID X Manager 2 Select the Size Standard tab 3 Select New 4 In the Size Standard Editor window Figure 12 select GeneMapper ID X Security Group as the Security Group This allows access for all users of the software Other security groups may be used 5 Enter a detailed name such as CC5_ILS_500_Y23_IDX 6 Choose Orange for the Size Standard Dye 7 Enter the sizes of the internal lane standard fragments 60 65 80 100 120 140 160 180 200 225 250 275 300 325 350 375 400 425 450 475 and 500 bases See Section 9 D Figure 24 8 Select OK 8257TA Figure 12 The GeneMapper ID X Software Version 1 2 Size Standard Editor Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 31 www promega com TMD035 Revised 3 15 6 D Creating a Casework Analysis Method with GeneMapper ID X Software Version 1 2 These instructions are intended as a guide to start analyzing data in GeneMapper ID X software They ar
6. Number of Reactions Final Volume Water Ampli cation Grade 17 5 l PowerPlex Y23 5X Master Mix 5 0 l PowerPlex Y23 10X Primer Pair Mix 2 5 l total reaction volume 25 l 1Add Water Ampli cation Grade to the tube rst and then add PowerPlex Y23 5X Master Mix and PowerPlex Y23 10X Primer Pair Mix For FTA card punches the template DNA will be added at Step 6 5 Vortex the PCR ampli cation mix for 5 10 seconds and then pipet 25 l of PCR ampli cation mix into each reaction well Failure to vortex the PCR ampli cation mix su ciently can result in poor ampli cation or locus to locus imbalance 6 For FTA storage cards add one or two 1 2mm punches from a card containing buccal cells or one 1 2mm punch from a card containing whole blood to the appropriate wells of the reaction plate For nonFTA card punches add the PCR ampli cation mix to the plate containing the PunchSolution Reagent treated punches Note It also is acceptable to add the FTA card punch rst then add the PCR ampli cation mix Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 11 www promega com TMD035 Revised 3 15 7 For the positive ampli cation control vortex the tube of 2800M Control DNA dilute an aliquot to 5 0ng l and add 1 l to a reaction well containing 25 l of
7. nal extension time Peak height imbalance Excess DNA in the ampli cation reaction can result in locus to locus imbalance within a dye channel such that the peak heights at the smaller loci are greater than those at the larger loci ski slope e ect Use less swab extract or reduce cycle number The cycle number was too high Decrease cycle number by one cycle and repeat the ampli cation Active SwabSolution Reagent carried over from swab extracts into the ampli cation reaction Larger loci are most susceptible to reagent carryover and will drop out before the smaller loci Ensure that the heat block reached 70 C 90 C if using 2 2ml Square Well Deep Well Plates and samples were incubated for the full 30 minutes Incubation for shorter time periods may result in incomplete reagent inactivation Do not use an incubator to incubate tubes or plates heat transfer is ine cient and will result in poor performance Use only a heat block to maintain e cient heat transfer Inactive SwabSolution Reagent Thaw the SwabSolution Reagent completely in a 37 C water bath and mix by gentle inversion Store the SwabSolution Reagent at 2 10 C Do not store reagents in the refrigerator door where the temperature can uctuate Do not re freeze as this may reduce activity DNA was not accessible on nonlytic material Small loci may amplify preferentially with large loci dropping out Pretreat swabs with SwabSolu
8. ABI PRISM 3100 or 3100 Avant Genetic Analyzer with POP 4 Polymer and Data Collection Software Version 2 0 Section 5 B GeneMapper ID Software Version 3 2 Figure 1 An overview of the PowerPlex Y23 System protocol 4 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD035 Revised 3 15 www promega com 2 Product Components and Storage Conditions P R O D U C T S I Z E C AT PowerPlex Y23 System 50 reac ons DC2305 Not For Medical Diagnostic Use This system contains su cient reagents for 50 reactions of 25 l each Includes Pre ampli cation Components Box 250 l PowerPlex Y23 5X Master Mix 125 l PowerPlex Y23 10X Primer Pair Mix 25 l 2800M Control DNA 10ng l 1 250 l Water Ampli cation Grade Post ampli cation Components Box 25 l PowerPlex Y23 Allelic Ladder Mix 300 l CC5 Internal Lane Standard 500 Y23 P R O D U C T S I Z E C AT PowerPlex Y23 System 200 reac ons DC2320 Not For Medical Diagnostic Use This system contains su cient reagents for 200 reactions of 25 l each Includes Pre ampli cation Components Box 4 250 l PowerPlex Y23 5X Master Mix 4 125 l PowerPlex Y23 10X Primer Pair Mix 25 l 2800M Control DNA 10ng l 5 1 250 l Water Ampli cation Grade Post ampli cation Components Box
9. File then New Project 2 Select Edit then Add Samples to Project 3 Browse to the location of run les Highlight desired les and then select Add to list followed by Add 4 In the Sample Type column use the drop down menu to select Allelic Ladder Sample Positive Control or Negative Control as appropriate for the sample Every folder in the project must contain at least one allelic ladder injection that is designated as Allelic Ladder in the Sample Type column for proper genotyping Note The positive control DNA de ned in the GeneMapper ID X panel le is the 2800M Control DNA Rede ne the genotype in the panel le if using a di erent positive control DNA 5 In the Analysis Method column select the analysis method created previously in this section 6 In the Panel column select the panels text le that was imported in Section 6 A 7 In the Size Standard column select the size standard that was imported in Section 6 B or created in Section 6 C 8 Select Analyze green arrow button to start data analysis Note By default the software is set to display the Analysis Requirements Summary window and Allelic Ladder Analysis Summary window if an issue is detected After analysis is complete the default setting is to show the Analysis Summary tab If these default settings are changed manual troubleshooting may be necessary 9
10. If all analysis requirements are met the Save Project window will open Figure 15 10 Enter the project name 11 Choose the applicable security group from the drop down menu and then select OK When the analysis is nished the Analysis Summary screen will appear We recommend that you review any yellow or red marker header bars in the plots view and handle them according to laboratory standard operating procedures The values displayed in the Analysis Method Peak Quality and SQ amp GQ Settings tabs are defaults and will a ect the quality values displayed in the plot settings We recommend that you modify the values in these tabs to t your laboratory s data analysis protocols Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 37 www promega com TMD035 Revised 3 15 6 F Importing PowerPlex Y23 Panels and Bins Text Files with GeneMapper ID Software Version 3 2 The instructions in this section were written using GeneMapper ID software version 3 2 Due to potential di erences between individual software versions some of the instructions may not apply to other software versions To facilitate analysis of data generated with the PowerPlex Y23 System we have created panels and bins text les to allow automatic assignment of genotypes using GeneMapper ID software version 3 2 We recommend that users of Gen
11. cards and PunchSolution Reagent Decreasing the reaction volume may result in suboptimal performance DNA was not accessible on nonlytic material Pretreat nonFTA materials with PunchSolution Reagent to ensure that DNA is liberated from cellular proteins Poor sample deposition Shedding and collection of donor cells was variable Increase cycle number Poor sample transfer to storage card or variable sampling from storage card Take punches from a di erent portion of the card Increasing cycle number can improve low peak heights Too much sample in the reaction Use one or two 1 2mm storage card punches see Section 4 B Follow the manufacturer s recommendations when depositing sample onto the storage card With storage cards reducing the reaction volumes below 25 l may result in ampli cation failure 54 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD035 Revised 3 15 www promega com 7 C Direct Ampli cation of DNA From Storage Card Punches continued Symptoms Causes and Comments Faint or absent allele peaks continued Ampli cation was inhibited when using more than one storage card punch with blood Use only one 1 2mm storage card punch with blood Active PunchSolution Reagent carried over into ampli cation reactions with nonFTA card punches Ensure that the heat block reached 70 C and samples we
12. Ampli cation of Extracted DNA 6 4 B Direct Ampli cation of DNA from Storage Card Punches 9 4 C Direct Ampli cation of DNA from Swabs 12 5 Instrument Setup and Sample Preparation 15 5 A Detection of Ampli ed Fragments Using the Applied Biosystems 3500 or 3500xL Genetic Analyzer with 3500 Data Collection Software Version 1 0 15 5 B Detection of Ampli ed Fragments Using POP 4 Polymer and the ABI PRISM 3100 or 3100 Avant Genetic Analyzer with Data Collection Software Version 2 0 or the Applied Biosystems 3130 or 3130xl Genetic Analyzer with Data Collection Software Version 3 0 26 6 Data Analysis 28 6 A Importing PowerPlex Y23 Panels Bins and Stutter Text Files into GeneMapper ID X Software Version 1 2 28 6 B Importing the CC5 ILS 500 IDX Size Standard into GeneMapper ID X Software Version 1 2 29 6 C Creating a Size Standa
13. DYS389II Fluorescein 259 303 24 35 DYS19 Fluorescein 312 352 9 19 DYS391 JOE 86 130 5 16 DYS481 JOE 139 184 17 32 DYS549 JOE 198 238 7 17 DYS533 JOE 245 285 7 17 DYS438 JOE 293 343 6 16 DYS437 JOE 344 380 11 18 DYS570 TMR ET 90 150 10 25 DYS635 TMR ET 150 202 15 28 DYS390 TMR ET 207 255 17 29 DYS439 TMR ET 263 307 6 17 DYS392 TMR ET 314 362 4 20 DYS643 TMR ET 368 423 6 17 DYS393 CXR ET 101 145 7 18 DYS458 CXR ET 159 215 10 24 DYS385a b CXR ET 223 307 7 28 DYS456 CXR ET 316 364 11 23 Y GATA H4 CXR ET 374 414 8 18 1The length of each allele in the allelic ladder has been con rmed by sequence analysis 2When using an internal lane standard such as the CC5 Internal Lane Standard 500 Y23 the calculated sizes of allelic ladder components may di er from those listed This occurs because di erent sequences in allelic ladder and ILS components may cause di erences in migration The dye label also a ects 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 and the Y Chromosome Haplotype Reference Database at www yhrd org Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in
14. To create a new File Name Convention Figure 7 navigate to the Library Select File Name Conventions and then select Create Alternatively a previously created File Name Convention may be used Select the File Name Attributes according to your laboratory practices and save with a descriptive name 9252TA Figure 7 The Create New File Name Convention window Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 23 www promega com TMD035 Revised 3 15 f To create a new Results Group Figure 8 navigate to the Library Select Results Group and then select Create Alternatively a previously created Results Group may be used Select the Results Group Attributes according to your laboratory practices Save with a descriptive name 9253TA Figure 8 The Create New Results Group window 24 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD035 Revised 3 15 www promega com 5 A Detection of Ampli ed Fragments Using the Applied Biosystems 3500 or 3500xL Genetic Analyzer with 3500 Data Collection Software Version 1 0 continued 3 To create a New Plate navigate to the Library and from the Manage menu select Plates then Create 4 Assign a descriptive plate name Select the plate type HID fro
15. appropriate septa Notes 1 Instrument detection limits vary therefore injection time or the amount of product mixed with loading cocktail may need to be adjusted To modify the injection time in the run module select Instrument Protocol from the Library menu in the data collection software If peak heights are higher than desired use less DNA template in the ampli cation reactions or reduce the number of cycles in the ampli cation program to achieve the desired signal intensity If the injection time is reduced a decreased peak amplitude threshold for the orange channel may be required for proper sizing 2 Use a volume of allelic ladder that results in peak heights that are all consistently above the peak amplitude threshold determined as part of your internal validation 6 Centrifuge the plate brie y to remove air bubbles from the wells 7 Denature samples at 95 C for 3 minutes then immediately chill on crushed ice or a freezer plate block or in an ice water bath for 3 minutes Denature samples just prior to loading the instrument Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 17 www promega com TMD035 Revised 3 15 Instrument Preparation Refer to the Applied Biosystems 3500 3500xL Genetic Analyzer User Guide for the instrument maintenance schedule and instructions to install the capillary array bu ers and p
16. 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 Alleles of STR loci are di erentiated by the number of copies of the repeat sequence contained within the ampli ed region and are distinguished from one another using uorescence detection following electrophoretic separation STR markers on the Y chromosome Y STR have qualities that are distinct from autosomal markers and are useful for human identi cation 10 16 Y STR markers are found on the nonrecombining region of the Y chromosome NRY and produce a haploid pro le when ampli ed from male DNA This quality simpli es male female mixture interpretation by removing the female contribution from an ampli cation pro le 17 18 Strict paternal inheritance of these markers makes them useful for paternity and kinship studies The PowerPlex Y23 System a d allows co ampli cation and four color uorescent detection of 23 loci including DYS576 DYS389I DYS448 DYS389II DYS19 DYS391 DYS481 DYS549 DYS533 DYS438 DYS437 DYS570 DYS635 DYS390 DYS439 DYS392 DYS643 DYS393 DYS458 DYS385a b DYS456 and Y GATA H4 The PowerPlex Y23 System is compatible with the ABI PRISM 3100 and 3100 Avant Genetic Analyzers and Applied
17. 6 Select the Allele tab Figure 21 7 Select the bins text le that was imported in Section 6 F 8 Ensure that the Use marker speci c stutter ratio if available box is checked Ensure that the appropriate global lter is applied to this analysis method For example for a 20 lter enter 0 20 for the Global Cut o Value for Tri Tetra and Penta repeats Figure 21 Note If you do not check the Use marker speci c stutter ratio if available box you will need to optimize these settings In house validation should be performed Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 43 www promega com TMD035 Revised 3 15 9 Enter the values shown in Figure 21 for proper ltering of peaks when using the PowerPlex Y23 System For an explanation of the proper usage and e ect of these settings refer to the Applied Biosystems user bulletin titled Installation Procedures and New Features for GeneMapper ID Software 3 2 10021TA Figure 21 The GeneMapper ID Software Version 3 2 Allele tab with settings for using a 20 peak lter 10 Select the Peak Detector tab We recommend the settings shown in Figure 20 Notes 1 Select full range or partial range for the analysis range When using a partial range choose an appropriate analysis range based on your data Choose a start point after the p
18. 9526 608 274 4330 Fax 608 277 2516 53 www promega com TMD035 Revised 3 15 Symptoms Causes and Comments Peak height imbalance Excessive amount of DNA Ampli cation of gt 0 5ng of template can result in an imbalance with smaller loci showing more product than larger loci Use less template or fewer cycles Degraded DNA sample DNA template was degraded and larger loci showed diminished yield Insu cient template DNA Use the recommended amount of template DNA if available Stochastic e ects can occur when amplifying low amounts of template The reaction volume was too low This system is optimized for a nal reaction volume of 25 l to overcome inhibitors present in DNA samples Decreasing the reaction volume can result in suboptimal performance Impure template DNA Inhibitors that may be present in forensic samples can lead to allele dropout or imbalance Imbalance may be seen more often when using the maximum template volume or a reduced ampli cation reaction volume 7 C Direct Ampli cation of DNA From Storage Card Punches The following information is speci c to direct ampli cation of DNA from storage card punches For additional information about general ampli cation and detection see Section 7 A Symptoms Causes and Comments Faint or absent allele peaks The reaction volume was too low This system is optimized for a nal reaction volume of 25 l to overcome inhibitors present in FTA
19. New Select Regular in the Type drop down list and select HIDFragmentAnalysis36_POP4 in the Template drop down list Con rm that the injection time is 5 seconds the injection voltage is 3kV and the run time is 1 500 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 G5 in the dye set drop down list Select OK 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 creat
20. USA 800 356 9526 608 274 4330 Fax 608 277 2516 69 www promega com TMD035 Revised 3 15 Table 8 The PowerPlex Y23 System Allele Determinations in Commonly Available Standard DNA Templates STR Locus Standard DNA Templates 2800M 99481 DYS5762 18 18 DYS389I 14 14 DYS4482 19 19 DYS389II 31 31 DYS19 14 14 DYS391 10 10 DYS481 22 22 DYS549 13 13 DYS533 12 12 DYS438 9 9 DYS437 14 14 DYS570 17 17 DYS635 21 21 DYS390 24 24 DYS439 12 12 DYS392 13 13 DYS643 10 10 DYS393 13 13 DYS458 17 17 DYS385a b 13 16 13 16 DYS456 17 17 Y GATA H4 11 11 1Information on strain 9948 is available online at http ccr coriell org Sections Search Sample_Detail aspx Ref GM09948 Information about the use of 9948 DNA as a standard DNA template can be found in reference 32 2A deletion has been reported at the DYS448 locus 33 Samples with this deletion will show two peaks i e duplication in DYS576 and a null allele in DYS448 70 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD035 Revised 3 15 www promega com 9 B Detection of Ampli ed Fragments Using the Applied Biosystems 3130 or 3130xl Genetic Analyzer with POP 7 Polymer and Data Collection Software Version 3 0 The PowerPlex Y23 System is optimized for POP 4 polymer We recogni
21. additional Y STR loci are included in this multiplex DYS481 DYS533 DYS549 DYS570 DYS576 and DYS643 were selected for their high genetic diversity 25 29 Table 8 lists the PowerPlex Y23 System alleles ampli ed from commonly available standard DNA templates Terminal nucleotide addition 34 35 occurs when a thermostable nonproofreading DNA polymerase adds a nucleo tide generally adenine to the 3 ends of ampli ed DNA fragments in a template independent manner The e ciency with which this occurs varies with di erent primer sequences Thus an artifact band one base shorter than expected i e missing the terminal addition is sometimes seen We have modi ed primer sequences and added a nal extension step at 60 C 36 to the ampli cation protocols to provide conditions for essentially complete terminal nucleotide addition when recommended amounts of template DNA are used Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 67 www promega com TMD035 Revised 3 15 Table 6 The PowerPlex Y23 System Locus Speci c Information STR Locus Label Chromosomal Location1 Repeat Sequence2 5 3 DYS576 Fluorescein Y AAAG DYS389I II Fluorescein Y TCTG TCTA DYS448 Fluorescein Y AGAGAT DYS19 Fluorescein Y TAGA DYS391 JOE Y TCTA DYS481 JOE Y CTT DYS549 JOE Y GATA DYS533 JOE Y ATC
22. and above the true allele peak respectively 2These variably sized peaks on the Applied Biosystems 3130 and 3500 Genetic Analyzers may represent double stranded DNA derived from the DYS448 amplicon Double stranded DNA is known to migrate faster than single stranded DNA on capillary electrophoresis CE instruments 3The low level DNA dependent artifact is noticeable only with high input template amounts and allele peak heights 4Artifact is observed more often with samples that contain relatively higher levels of female DNA 48 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD035 Revised 3 15 www promega com 6 L Results continued Table 5 DNA Independent Artifacts Observed with the PowerPlex Y23 System Dye Label Artifact Size Fluorescein 68 71 bases1 66 69 bases1 138 145 bases2 JOE 60 62 bases1 58 60 bases1 138 145 bases2 1The signal strength of these artifacts increases with storage of the ampli cation plate at 4 C sometimes in as short a time period as overnight but more commonly when plates are left at 4 C for a few days We recommend storing ampli cation products at 20 C 2Artifact may appear as a dye blob or a peak in sample reaction and negative control reaction 7 Troubleshooting For questions not addressed here please contact your local Promega Branch O ce or Dist
23. and condi ons Restric ons on Use End Users are speci cally not authorized to and are forbidden from reselling transferring or distribu ng any products either as a stand alone product or as a component of another product The right to use the products does not in and of itself include or carry any right of the End User to any GE Healthcare Bio Sciences Corp s technology or intellectual property other than expressly provided herein End Users may not use sequence s in an a empt to reverse engineer parameters of any of GE Healthcare Bio Sciences Corp proprietary products or services Disclaimer of Warran es GE Healthcare Bio Sciences Corp provides no warran es to end user statutory or implied including without limita on as to product quality condi on descrip on merchantability or tness for a par cular purpose and all such warran es are hereby expressly disclaimed GE Healthcare Bio Sciences Corp hereby expressly disclaims any warranty regarding results obtained through the use of the products including without limita on any claim of inaccurate invalid or incomplete results Exclusion of Liability GE Healthcare Bio Sciences Corp and its a liates shall have no liability to an End User including without limita on for any loss of use or pro ts business interrup on or any consequen al incidental special or other indirect damages of any kind regardless of how caused and regardless of whether an ac on in contract tort str
24. chosen in Section 6 D or 6 E Adjust the starting data point in the analysis method Alternatively use a full range for the analysis Extra peaks in 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 CC5 ILS 500 Y23 peaks de ned 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 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 61 www promega com TMD035 Revised 3 15 Symptoms Causes and Comments Peaks in size standard missing If peaks are below threshold decrease the peak amplitude threshold in the analysis method for the orange channel to include peaks or increase the volume of CC5 ILS 500 Y23 used in Section 5 If peaks are low quality rede ne the size standard for the sample to skip these peaks An incorrect size standard was used Signi cantly raised baseline Poor spectral calibration Perform a new spectral calibration and re run the samples Incorrect G5 spectral was active Re run samples and con rm that the PowerPlex 5 dye G5 spectral is set for G5 See instructions for instrument preparation in Sec
25. data collection software to modify the injection time or voltage in the run module see Instrument Preparation below If the injection time or voltage is reduced a decreased peak amplitude threshold for the orange channel may be required for proper sizing 6 Centrifuge plate brie y to remove air bubbles from the wells 7 Denature samples at 95 C for 3 minutes then immediately chill on crushed ice or a freezer plate block or in an ice water bath for 3 minutes Denature samples just prior to loading the instrument 72 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD035 Revised 3 15 www promega com 9 B Detection of Ampli ed Fragments Using the Applied Biosystems 3130 or 3130xl Genetic Analyzer with POP 7 Polymer and Data Collection Software Version 3 0 continued Instrument Preparation Refer to the instrument user s 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 Applied Biosystems 3130 or 3130xl Genetic Analyzer with Data Collection Software Version 3 0 with the following exceptions 1 In the Module Manager select New Select Regular in the Type drop down list and select FragmentAnalysis36_POP7 in the Template drop down list Con rm that the in
26. of STR ampli cation Ampli cation of STRs 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 a 20 minute extension step at 60 C after thermal cycling Section 4 Decrease the amount of template DNA Using more than the recommended amount of template DNA can result in incomplete adenylation Decrease cycle number Increase the nal extension time Artifacts The signal strength of certain artifacts increases with storage of the ampli cation plate at 4 C see Table 5 sometimes in as short a time period as overnight but more commonly when plates are left at 4 C for a few days We recommend storing ampli cation products at 20 C 50 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD035 Revised 3 15 www promega com 7 A Ampli cation and Fragment Detection continued Symptoms Causes and Comments Extra peaks visible in one CE related artifacts spikes Minor voltage changes or urea or all color channels continued crystals passing by the laser can cause spikes or unexpected peaks Spikes sometimes appear in one color but often are easily identi ed by their presence in more than one color Re inject samples to con rm CE related artifacts contaminants Contaminants in the water used with
27. or solutions The DNA IQ System eliminates PCR inhibitors and contaminants frequently encountered in casework samples In additional DNA has been isolated from casework samples such as tissue di erentially separated sexual assault samples and stains on support materials The DNA IQ System has been tested with PowerPlex Systems to ensure a streamlined process For applications requiring human speci c DNA quanti cation the Plexor HY System Cat DC1000 was developed 38 For information about automation of Promega chemistries on automated workstations using Identity Automation solutions contact your local Promega Branch O ce or Distributor contact information available at www promega com support worldwide contacts e mail genetic promega com or visit www promega com idautomation 74 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD035 Revised 3 15 www promega com 9 D The CC5 Internal Lane Standard 500 Y23 The CC5 Internal Lane Standard 500 Y23 contains 21 DNA fragments of 60 65 80 100 120 140 160 180 200 225 250 275 300 325 350 375 400 425 450 475 and 500 bases in length Figure 24 Each fragment is labeled with CC5 dye and can be detected separately as a fth color in the presence of PowerPlex Y23 ampli ed material The CC5 ILS 500 Y23 is designed for use in each CE injection to in
28. panels and bin sets Matrix standards are required for initial setup of the color separation matrix The matrix standards are available separately for ABI PRISM 3100 and 3100 Avant Genetic Analyzers and Applied Biosystems 3130 3130xl 3500 and 3500xL Genetic Analyzers PowerPlex 5 Dye Matrix Standards 3100 3130 Cat DG4700 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 19 20 Guidelines for the validation process are published in the Internal Validation Guide of Y STR Systems for Forensic Laboratories 21 The quality of puri ed DNA or direct ampli cation samples quality of plasticware small changes in bu ers ionic strength primer concentrations choice of thermal cycler and thermal cycling conditions can a ect PCR success We suggest strict adherence to recommended procedures for ampli cation and uorescence detection Additional research and validation are required if any modi cations are made to the recommended protocols 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 template DNA handling primer pairs assembling ampli cation reactions and analyzing ampli cation products Reagents and materials used prior to ampli cation PowerPle
29. reaction Liquid blood from collection or storage Vacutainer tubes or nger sticks spotted onto FTA cards one punch per 25 l ampli cation reaction NonFTA sample types include Buccal samples on Bode Buccal DNA Collector devices one punch per 25 l ampli cation reaction Blood and buccal samples on nonFTA cards e g S amp S 903 one punch per 25 l ampli cation reaction Pretreat nonFTA sample types with the PunchSolution Kit Cat DC9271 to lyse nonFTA samples before adding the PCR ampli cation mix For more information see the PunchSolution Kit Technical Manual TMD038 Failure to pretreat these samples may result in incomplete pro les Use a manual punch tool with a 1 2mm tip to manually create sample disks from a storage card Place tip near the center of the sample spot and with a twisting or pressing action cut a 1 2mm sample disk Use the plunger to eject the disk into the appropriate well of a reaction plate Automated punchers also can be used to create sample disks Refer to the user s guide for your instrument for assistance with generating 1 2mm disks technical advice and troubleshooting information Note Static may be problematic when adding a punch to a well For FTA card punches adding PCR ampli cation mix to the well before adding the punch may help alleviate static problems For nonFTA card punches adding PunchSolution Reagent to the well before adding th
30. the instrument or to dilute the 10X genetic analyzer bu er may generate peaks in the uorescein and JOE channels Use autoclaved deionized water change vials and wash bu er reservoir An incorrect internal lane standard was used Be sure to use the CC5 ILS 500 Y23 as the size standard when using the PowerPlex Y23 System Do not use the CC5 ILS 500 Cat DG1521 The CC5_ILS_500 xml or CC5_ILS_500_IDX xml le can be used to assign fragment sizes for the CC5 ILS 500 Y23 Incorrect G5 spectral was active when analyzing samples with the Applied Biosystems 3130 or 3130xl Genetic Analyzer Re run samples and con rm that the PowerPlex 5 dye spectral is set for G5 See instructions for instrument preparation in Section 5 The wrong spectral calibration was used Make sure that the spectral calibration was performed using the same polymer type as that for sample analysis e g do not use a POP 4 generated spectral calibration for a POP 7 run Pull up or bleedthrough Pull up can occur when peak heights are too high or if a poor or incorrect matrix is applied to the samples Perform a new spectral calibration and re run the samples Instrument sensitivities can vary Optimize the injection conditions See Section 5 Reboot the Applied Biosystems 3500 or 3500xL Genetic Analyzer and the instrument s computer Repeat the spectral calibration Do not allow borrowing when running the spectral calibration on
31. the protocol detailed below Ampli cation Setup 1 Thaw the PowerPlex Y23 5X Master Mix PowerPlex Y23 10X Primer Pair Mix and Water Ampli cation Grade completely Note Centrifuge tubes brie y to bring contents to the bottom and then vortex reagents for 15 seconds before each use Do not centrifuge the 10X Primer Pair Mix or 5X Master Mix after vortexing as this may cause the reagents 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 ampli cation mix for all samples It also ensures that each reaction contains the same PCR ampli cation mix 3 Use a clean MicroAmp plate for reaction assembly and label appropriately Alternatively determine the number of clean 0 2ml reaction tubes required and label appropriately Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 7 www promega com TMD035 Revised 3 15 4 Add the nal volume of each reagent listed in Table 1 to a sterile tube Table 1 PCR Ampli cation Mix for Ampli cation of Extracted DNA PCR Ampli cation Mix Component1 Volume Per Reaction Number of Reactio
32. 0 Fax 608 277 2516 TMD035 Revised 3 15 www promega com 4 Protocols for DNA Ampli cation Using the PowerPlex Y23 System The PowerPlex Y23 System is optimized for the GeneAmp PCR System 9700 thermal cycler The use of gloves and aerosol resistant pipette tips is highly recommended to prevent cross contamination Keep all pre ampli cation and post ampli cation reagents in separate rooms Prepare ampli cation reactions in a room dedicated for reaction setup Use equipment and supplies dedicated for ampli cation setup Meticulous care must be taken to ensure successful ampli cation A guide to ampli cation troubleshooting is provided in Section 7 The concentration of 2800M Control DNA was determined by measuring absorbance at 260nm Quanti cation of this control DNA by other methods such as qPCR may result in a di erent value Prepare a fresh DNA dilution for each set of ampli cations Do not store diluted DNA e g 0 25ng l or less 4 A Ampli cation of Extracted DNA Materials to Be Supplied by the User GeneAmp PCR System 9700 thermal cycler with a gold plated or silver plated sample block Applied Biosystems centrifuge compatible with a 96 well plate or reaction tubes MicroAmp optical 96 well reaction plate or 0 2ml MicroAmp reaction tubes Applied Biosystems aerosol resistant pipette tips We routinely amplify 0 5ng of template DNA in a 25 l reaction volume using
33. 30 Fax 608 277 2516 75 www promega com TMD035 Revised 3 15 9 F Related Products STR Systems Product Size Cat PowerPlex Fusion 6C System 50 or 100 direct amp reactions DC2705 200 or 400 direct amp reactions DC2720 PowerPlex Fusion System 200 reactions DC2402 800 reactions DC2408 PowerPlex ESX 16 Fast System 100 reactions DC1611 400 reactions DC1610 PowerPlex ESX 17 Fast System 100 reactions DC1711 400 reactions DC1710 PowerPlex ESI 16 Fast System 100 reactions DC1621 400 reactions DC1620 PowerPlex ESI 17 Fast System 100 reactions DC1721 400 reactions DC1720 PowerPlex 21 System 200 reactions DC8902 4 200 reactions DC8942 PowerPlex 16 HS System 100 reactions DC2101 400 reactions DC2100 PowerPlex 18D System 200 reactions DC1802 800 reactions DC1808 PowerPlex S5 System 100 reactions DC6951 400 reactions DC6950 PowerPlex CS7 System 100 reactions DC6613 Not for Medical Diagnostic Use Accessory Components Product Size Cat PowerPlex 5 Dye Matrix Standards 3130 3130 25 l each dye DG4700 CC5 Internal Lane Standard 500 Y23 300 l DG3801 2800M Control DNA 10ng l 25 l DD7101 2800M Control DNA 0 25ng l 500 l DD7251 PunchSolution Kit 100 preps DC9271 SwabSolution Kit 100 preps DC8271 Water Ampli cation Grade 6 250 l 5 1 250 l DW0991 76 Promega Corpora on 2800
34. 5ng of DNA template The amount of DNA template used in your laboratory should be based on the results of your internal validation and may be di erent 5 Vortex the PCR ampli cation mix for 5 10 seconds and then pipet PCR ampli cation mix into each reaction well or tube Failure to vortex the PCR ampli cation mix su ciently can result in poor ampli cation or locus to locus imbalance 6 Add template DNA for each sample to the respective well or tube containing PCR ampli cation mix Note The PowerPlex Y23 System is optimized and balanced for 0 5ng of DNA template The amount of DNA template used in your laboratory should be based on the results of your internal validation and may be di erent 7 For the positive ampli cation control vortex the tube of 2800M Control DNA and then dilute an aliquot to 0 5ng in the desired template DNA volume Add 0 5ng of diluted DNA to a reaction well or tube containing PCR ampli cation mix 8 For the negative ampli cation control pipet Water Ampli cation Grade or TE 4 bu er instead of template DNA into a reaction well containing PCR ampli cation mix 9 Seal or cap the plate or close the tubes Optional Brie y centrifuge the plate or tubes to bring contents to the bottom of the wells and remove any air bubbles 8 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax
35. 608 277 2516 TMD035 Revised 3 15 www promega com 4 A Ampli cation of Extracted DNA continued Thermal Cycling Ampli cation and detection instrumentation may vary You may need to optimize protocols including the amount of template DNA cycle number injection conditions and loading volume for your laboratory instrumentation Testing at Promega shows that 30 cycles works well for 0 5ng of puri ed DNA template 1 Place the MicroAmp plate or reaction tubes in the thermal cycler 2 Select and run the recommended protocol which is provided below Be sure to select Max Mode as the ramp speed This requires a silver plated or gold plated sample block The ramp speed is set after the thermal cycling run is started When the Select Method Options screen appears select Max for the ramp speed and enter the reaction volume The estimated total cycle time is 1 hour and 40 minutes Thermal Cycling Protocol 96 C for 2 minutes then 94 C for 10 seconds 61 C for 1 minute 72 C for 30 seconds for 30 cycles then 60 C for 20 minutes 4 C soak 3 After completion of the thermal cycling protocol proceed with fragment analysis or store ampli ed samples at 20 C in a light protected box Note Long term storage of ampli ed samples at 4 C or higher may produce artifacts Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 2
36. 7 2516 TMD035 Revised 3 15 www promega com 6 L Results continued 10610TA A B C D Figure 23 The PowerPlex Y23 Allelic Ladder Mix The PowerPlex Y23 Allelic Ladder Mix was analyzed using an Applied Biosystems 3130 Genetic Analyzer and a 3kV 3 second injection The sample le was analyzed with the GeneMapper ID software version 3 2 and PowerPlex Y23 panels and bins text les Panel A The uorescein labeled allelic ladder components and their allele designations Panel B The JOE labeled allelic ladder components and their allele designations Panel C The TMR ET labeled allelic ladder components and their allele designations Panel D The CXR ET labeled allelic ladder components and their allele designations Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 47 www promega com TMD035 Revised 3 15 Artifacts and Stutter Stutter products are a common ampli cation artifact associated with STR analysis 23 24 Stutter products often are 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 Trinucleotide repeat loci will generally exhibit higher stutter than loci with longer repeat lengths DYS481 is a trinucleotide repe
37. 77 2516 9 www promega com TMD035 Revised 3 15 4 B Direct Ampli cation of DNA from Storage Card Punches Materials to Be Supplied by the User GeneAmp PCR System 9700 thermal cycler with a gold plated or silver plated sample block Applied Biosystems centrifuge compatible with a 96 well plate MicroAmp optical 96 well reaction plate Applied Biosystems aerosol resistant pipette tips PunchSolution Kit Cat DC9271 for nonFTA card punches 5X AmpSolution Reagent for FTA card punches Cat DM1231 1 2mm Harris Micro Punch or equivalent manual punch and cutting mat or automated punch system This section contains a protocol for direct ampli cation of DNA from storage card punches using the PowerPlex Y23 System and GeneAmp PCR System 9700 thermal cycler When using the protocol detailed below add the number of 1 2mm storage card punches indicated below to each 25 l ampli cation reaction Note You will need to optimize and validate the number of storage card punches per reaction in your laboratory See the PCR Optimization recommendations at the end of this section FTA based sample types include Buccal cells collected on FTA cards with Whatman EasiCollect or Fitzco Sampact devices one or two punches per 25 l ampli cation reaction Buccal cells collected with swabs transferred to FTA or Indicating FTA cards one or two punches per 25 l ampli cation
38. Biosystems 3130 3130xl 3500 and 3500xL Genetic Analyzers Ampli cation and detection instrumentation may vary You may need to optimize protocols including the amount of template DNA cycle number injection conditions and loading volume for your laboratory instrumentation In house validation should be performed We have tested the PowerPlex Y23 System with Data Collection Software Versions 2 0 and 3 0 3500 Data Collection Software Version 1 0 GeneMapper ID X Software Version 1 2 and GeneMapper ID Software Version 3 2 Other software versions may be available for use however the options available in other versions may di er slightly from the options listed in this Technical Manual 7 Troubleshooting 48 7 A Ampli cation and Fragment Detection 48 7 B Ampli cation of Extracted DNA 52 7 C Direct Ampli cation of DNA From Storage Card Punches 53 7 D Direct Ampli cation of DNA From Swabs 56 7 E GeneMapper ID X Software
39. File then New Project 2 Select Edit then Add Samples to Project 3 Browse to the location of the run les Highlight desired les then select Add to list followed by Add 4 In the Sample Type column use the drop down menu to select Ladder Sample Positive Control or Negative Control as appropriate for the sample Every folder in the project must contain at least one allelic ladder injection that is designated as Ladder in the Sample Type column for proper genotyping Note The positive control DNA de ned in the GeneMapper ID X panel le is the 2800M Control DNA Rede ne the genotype in the panel le if using a di erent positive control DNA 5 In the Analysis Method column select the analysis method created previously in this section 6 In the Panel column select the panels text le that was imported in Section 6 F 7 In the Size Standard column select the size standard that was imported in Section 6 G or created in Section 6 H 8 Select Analyze green arrow button to start the data analysis 6 K Controls 1 Observe the results for the negative control Using the protocols de ned in this manual the negative control should be devoid of ampli cation products 2 Observe the results for the 2800M Control DNA The expected 2800M DNA allele designations for each locus are listed in Table 8 Section 9 A Promega Co
40. PCR ampli cation mix Notes 1 Optimization of the amount of 2800M Control DNA may be required depending on thermal cycling conditions and laboratory preferences 2 Do not include blank storage card punches in the positive control reactions 8 Reserve a well containing PCR ampli cation mix as a negative ampli cation control Note An additional negative control with a blank punch may be performed to detect contamination from the storage card or punch device 9 Seal or cap the plate Brie y centrifuge the plate to bring storage card punches to the bottom of the wells and remove air bubbles Thermal Cycling Ampli cation and detection instrumentation may vary You will need to optimize protocols including the number of storage card punches cycle number injection conditions and loading volume for each laboratory instrument Testing at Promega shows that 26 cycles works well for a variety of sample types Buccal samples may require more ampli cation cycles than blood samples Cycle number will need to be optimized in each laboratory for each sample type that is ampli ed see below 1 Place the MicroAmp plate in the thermal cycler 2 Select and run the recommended protocol which is provided below Be sure to select Max mode as the ramp speed This requires a silver plated or gold plated sample block The ramp speed is set after the thermal cycling run is started When the Select Method Options screen appear
41. POP 7 speci c panels and bins text les for GeneMapper and GeneMapper ID X software Materials to Be Supplied by the User 95 C dry heating block water bath or thermal cycler crushed ice ice water bath or a freezer plate block centrifuge compatible with 96 well plates aerosol resistant pipette tips 3130 or 3103xl capillary array 36cm plate retainer and base set standard POP 7 polymer for the Applied Biosystems 3130 Genetic Analyzer 10X genetic analyzer bu er with EDTA MicroAmp optical 96 well plate or equivalent and septa Hi Di formamide Applied Biosystems Cat 4311320 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 71 www promega com TMD035 Revised 3 15 The quality of formamide is critical Use Hi Di formamide Freeze formamide in aliquots at 20 C Multiple freeze thaw cycles or long term storage at 4 C may cause breakdown of formamide Poor quality formamide 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 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 formami
42. Revised 3 15 TMD035 T E C H N I C A L M A N U A L PowerPlex Y23 System Instruc ons for Use of Products DC2305 and DC2320 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 1 www promega com TMD035 Revised 3 15 All technical literature is available at www promega com protocols Visit the web site to verify that you are using the most current version of this Technical Manual E mail Promega Technical Services if you have questions on use of this system genetic promega com PowerPlex Y23 System 1 Description 2 2 Product Components and Storage Conditions 4 3 Before You Begin 5 3 A Precautions 5 3 B Spectral Calibration 5 4 Protocols for DNA Ampli cation Using the PowerPlex Y23 System 6 4 A
43. T DYS438 JOE Y TTTTC DYS437 JOE Y TCTA DYS570 TMR ET Y TTTC DYS635 TMR ET Y TSTA compound DYS390 TMR ET Y TCTA TCTG DYS439 TMR ET Y AGAT DYS392 TMR ET Y TAT DYS643 TMR ET Y CTTTT DYS393 CXR ET Y AGAT DYS458 CXR ET Y GAAA DYS385a b CXR ET Y GAAA DYS456 CXR ET Y AGAT Y GATA H4 CXR ET Y TAGA 1Information about most of these loci can be found at www cstl nist gov biotech strbase chrom htm 2The August 1997 report 30 31 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 de ned using the rst possible 5 nucleotide of a repeat motif and 2 for STR loci not associated with a coding gene the rst database entry or original literature description shall be used 68 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD035 Revised 3 15 www promega com 9 A Advantages of Using the Loci in the PowerPlex Y23 System continued Table 7 The PowerPlex Y23 System Allelic Ladder Information STR Locus Label Size Range of Allelic Ladder Components1 2 bases Repeat Numbers of Allelic Ladder Components3 DYS576 Fluorescein 97 145 11 23 DYS389I Fluorescein 147 179 9 17 DYS448 Fluorescein 196 256 14 24
44. Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 27 www promega com TMD035 Revised 3 15 5 Add 1 l of ampli ed sample or 1 l of PowerPlex Y23 Allelic Ladder Mix to each well Cover wells with appropriate septa Note Instrument detection limits vary therefore injection time injection voltage or the amount of sample mixed with loading cocktail may need to be adjusted Use the Module Manager in the data collection software to modify the injection time or voltage in the run module see Instrument Preparation below If the injection time or voltage is reduced a decreased peak amplitude threshold for the orange channel may be required for proper sizing 6 Centrifuge plate brie y to remove air bubbles from the wells 7 Denature samples at 95 C for 3 minutes then immediately chill on crushed ice or a freezer plate block or in an ice water bath for 3 minutes Denature samples just prior to loading the instrument Instrument Preparation Refer to the instrument user s 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 Data Collection Software Version 3 0 with the following exceptions 1 In the Module Manager select
45. Values for peak amplitude thresholds are usually 50 150RFU for data generated using the ABI PRISM 3100 and 3100 Avant Genetic Analyzers and Applied Biosystems 3130 and 3130xl Genetic Analyzers However individual laboratories should determine their peak amplitude thresholds from internal validation studies Peak heights for the CC5 ILS 500 Y23 are generally lower than those for the other dyes Therefore the threshold for the orange dye may be lower than that for the other dyes 8187TA Figure 20 The GeneMapper ID Software Version 3 2 Peak Detector tab 11 Select the Peak Quality tab You may change the settings for peak quality Note See the GeneMapper ID user s manual for more information The settings in Steps 11 and 12 should be based on the results of your internal validation 12 Select the Quality Flags tab You may change these settings 13 Select OK to save your settings 42 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD035 Revised 3 15 www promega com 6 I Creating a Casework Analysis Method with GeneMapper ID Software Version 3 2 continued Processing Data for Casework Samples 1 Select File then New Project 2 Select Edit then Add Samples to Project 3 Browse to the location of the run les Highlight desired les then select Add to list followe
46. Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD035 Revised 3 15 www promega com 9 F Related Products continued Sample Preparation and DNA Quanti cation Systems Product Size Cat PowerQuant System 200 reactions PQ5002 800 reactions PQ5008 Plexor HY System 200 reactions DC1001 800 reactions DC1000 DNA IQ System 100 reactions DC6701 400 reactions DC6700 Not for Medical Diagnostic Use 9 G Summary of Changes The following changes were made to the 3 15 revision of this document 1 The document was updated to include information about compatibility of additional software versions update the newest compatible kits provide more detail to necessary testing materials and conditions provide updates to software gures to better represent instructions and update the Troubleshooting Section with additional symptoms causes and comments 2 The list of artifacts in Section 6 L was updated 3 The document design was updated Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 77 www promega com TMD035 Revised 3 15 a U S Pat No 6 242 235 Australian Pat No 761757 Canadian Pat No 2 335 153 Chinese Pat No ZL99808861 7 Hong Kong Pat No HK 1040262 Japanese Pat No 3673175 European Pat No 1088060 and other paten
47. X Primer Pair Mix and 5X Master Mix completely Vortex the 5X Primer Pair Mix and 5X Master Mix for 15 seconds before use do not centrifuge the 10X Primer Pair Mix or 5X Master Mix after mixing Calibrate thermal cyclers and pipettes routinely PCR ampli cation mix prepared in Section 4 was not mixed well Vortex the PCR ampli cation mix for 5 10 seconds before dispensing into the reaction tubes or plate Tubes of 5X Master Mix and 10X Primer Pair Mix from di erent lots were used The PowerPlex Y23 5X Master Mix and PowerPlex Y23 10X Primer Pair Mix are manufactured as a matched set for optimal performance If lots are mixed locus to locus imbalance and variation in signal intensity may occur 52 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD035 Revised 3 15 www promega com 7 B Ampli cation of Extracted DNA The following information is speci c to ampli cation of extracted DNA For information about general ampli cation and detection see Section 7 A Symptoms Causes and Comments Faint or absent allele peaks Impure template DNA Because a small amount of template is used this is rarely a problem Depending on the DNA extraction procedure used and sample source inhibitors might be present in the DNA sample Faint or absent peaks may be seen more often when using the maximum template volume or reduced amp
48. after pretreatment using the SwabSolution Kit For additional information about general ampli cation and detection see Section 7 A Symptoms Causes and Comments Faint or absent allele peaks Poor sample deposition Shedding and collection of donor cells was variable Increase cycle number Inactive SwabSolution Reagent Thaw the SwabSolution Reagent completely in a 37 C water bath and mix by gentle inversion Store the SwabSolution Reagent at 2 10 C Do not store reagents in the refrigerator door where the temperature can uctuate Do not refreeze as this may reduce activity Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 57 www promega com TMD035 Revised 3 15 Symptoms Causes and Comments Faint or absent allele peaks continued Active SwabSolution Reagent carried over into the ampli cation reaction Ensure that the heat block reached 70 C 90 C if using a 2 2ml Square Well Deep Well Plate and samples were incubated for the full 30 minutes Incubation for shorter time periods may result in incomplete reagent inactivation Do not use an incubator to incubate tubes or plates heat transfer is ine cient and will result in poor performance Use only a heat block to maintain e cient heat transfer We have tested 60 minute incubation times and observed no di erence in performance compared to a 30 minute i
49. at and exhibits exceptionally high stutter The pattern and intensity of stutter may di er slightly between primer sets for the same locus The mean plus three standard deviations at each locus is used in the PowerPlex Y23 panels text les for locus speci c ltering in the GeneMapper ID software version 3 2 and in the PowerPlex Y23 stutter text les for locus speci c ltering in GeneMapper ID X software The GeneMapper ID X stutter les also include lters for the plus stutter associated with the two trinucleotide repeat loci DYS481 and DYS392 as well as lters for the plus 2 and minus 2 base artifacts associated with the DYS19 locus In addition to stutter peaks DNA dependent artifact peaks Table 4 and DNA independent artifact peaks Table 5 may be observed with the PowerPlex Y23 System A low level artifact peak at approximately 172 bases may be observed with the CC5 ILS 500 Y23 in the orange channel The peak height of this artifact may vary from lot to lot and may be labeled by the software This peak is not used during sizing of the peaks present in the sample Table 4 DNA Dependent Artifacts Observed with the PowerPlex Y23 System Locus Artifact Size DYS19 n 2 n 21 DYS448 n 9 to n 152 3 DYS635 160 bases4 DYS481 164 bases4 DYS549 187 bases4 DYS458 201 bases4 DYS533 253 bases4 DYS533 272 bases4 DYS643 427 bases4 DYS643 440 bases4 1Two bases below
50. ax 608 277 2516 TMD035 Revised 3 15 www promega com 7 F GeneMapper ID Software continued Symptoms Causes and Comments O ladder alleles An allelic ladder from a di erent run than the samples was used Re analyze samples with an allelic ladder 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 I or 6 J Panels text le selected for analysis was incorrect for the STR system used Assign correct panels le that corresponds to the STR system used for ampli cation The allelic ladder was not identi ed 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 identi ed in the sample Manually rede ne the sizes of the size standard fragments in the sample A low quality allelic ladder was used during analysis Ensure that only high quality allelic ladders are used for analysis Size standard not called correctly Starting data point was incorrect for the partial range chosen in Section 6 I or 6 J 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 Edito
51. crease precision in analyses when using the PowerPlex Y23 System Protocols to prepare and use this internal lane standard are provided in Section 5 Be sure to use the CC5 ILS 500 Y23 as the size standard for the PowerPlex Y23 System Do not use the CC5 ILS 500 Cat DG1521 The CC5_ILS_500 xml le can be used to assign fragment sizes for the CC5 ILS 500 Y23 A low level artifact peak at approximately 172 bases may be observed with the CC5 ILS 500 Y23 in the orange channel The peak height of this artifact may vary from lot to lot and may be labeled by the software This peak is not used during sizing of the peaks present in the sample 10607TA Figure 24 CC5 Internal Lane Standard 500 Y23 An electropherogram showing the CC5 Internal Lane Standard 500 Y23 fragments 9 E Composition of Bu ers and Solutions TE 4 bu er 10mM Tris HCl 0 1mM EDTA pH 8 0 1 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 nal volume to 1 liter with deionized water TE 4 bu er with 20 g ml glycogen 1 21g Tris base 0 037g EDTA Na2EDTA 2H2O 20 g ml glycogen Dissolve Tris base and EDTA in 900ml of deionized water Adjust to pH 8 0 with HCl Add glycogen Bring the nal volume to 1 liter with deionized water Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 43
52. d Comments Peak height imbalance continued Active PunchSolution Reagent carried over into ampli cation reactions with nonFTA card punches Larger loci are most susceptible to carryover and will drop out before the smaller loci Ensure that the heat block reached 70 C and samples were incubated for 30 minutes or until wells are dry Incubation for shorter time periods may result in incomplete inactivation of the PunchSolution Reagent Using a smaller ampli cation reaction volume may compromise performance when using 10 l of PunchSolution Reagent Reducing the PunchSolution Reagent volume may improve results for reactions with reduced ampli cation volumes Optimization and validation are required Inactive PunchSolution Reagent was used to pretreat nonFTA punches Thaw the PunchSolution Reagent at 2 10 C Do not store reagents in the refrigerator door where the temperature can uctuate Do not refreeze as this may reduce activity Extreme variability in sample There can be signi cant individual to individual variability in the to sample peak heights number of cells on a punch resulting in peak height variability between samples The PunchSolution Kit maximizes the recovery of ampli able DNA from nonFTA punches but does not normalize the amount of DNA present 7 D Direct Ampli cation of DNA From Swabs The following information is speci c to direct ampli cation of DNA from swabs
53. d by Add 4 In the Sample Type column use the drop down menu to select Ladder Sample Positive Control or Negative Control as appropriate for the sample Every folder in the project must contain at least one allelic ladder injection that is designated as Ladder in the Sample Type column for proper genotyping Note The positive control DNA de ned in the GeneMapper ID X panel le is the 2800M Control DNA Rede ne the genotype in the panel le if using a di erent positive control DNA 5 In the Analysis Method column select the analysis method created previously in this section 6 In the Panel column select the panels text le that was imported in Section 6 F 7 In the Size Standard column select the size standard that was imported in Section 6 G or created in Section 6 H 8 Select Analyze green arrow button to start data analysis 6 J Creating a Databasing or Paternity Analysis Method Using a Global Filter with GeneMapper ID Software Version 3 2 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 PowerPlexY23_20 lter
54. de Sample Preparation 1 Thaw the CC5 Internal Lane Standard 500 Y23 and PowerPlex Y23 Allelic Ladder Mix Note Centrifuge tubes brie y to bring contents to the bottom then vortex for 15 seconds before each use Do not centrifuge after vortexing as this may cause the reagents to be concentrated at the bottom of the tube 2 Prepare a loading cocktail by combining and mixing CC5 ILS 500 Y23 and Hi Di formamide as follows 1 0 l CC5 ILS 500 Y23 samples 10 0 l Hi Di formamide samples Be sure to use the CC5 ILS 500 Y23 as the size standard when using the PowerPlex Y23 System Do not use the CC5 ILS 500 Cat DG1521 The CC5_ILS_500 xml le can be used to assign fragment sizes for the CC5 ILS 500 Y23 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 Keep the volume of formamide at 10 0 l per well and adjust the volume added to the wells in Step 4 accordingly 3 Vortex for 10 15 seconds to mix 4 Pipet 11 l of formamide internal lane standard mix into each well 5 Add 1 l of ampli ed sample or 1 l of PowerPlex Y23 Allelic Ladder Mix to each well Cover wells with appropriate septa Note Instrument detection limits vary therefore injection time injection voltage or the amount of product mixed with loading cocktail may need to be adjusted Use the Module Manager in the
55. e Applied Biosystems user bulletin titled Installation Procedures and New Features for GeneMapper ID Software 3 2 Notes 1 The PowerPlex Y23 System includes two trinucleotide repeat loci DYS481 and DYS392 Both of these loci exhibit plus stutter The plus stutter lter of 0 06 will lter the majority of the plus stutter for DYS481 but not for DYS392 A lter value of 0 1 is needed to lter most of the plus stutter for DYS392 2 Some of these settings have been optimized and are di erent from the recommended settings in the user bulletin You may need to optimize these settings In house validation is required 10020TA Figure 19 The GeneMapper ID Software Version 3 2 Allele tab Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 41 www promega com TMD035 Revised 3 15 10 Select the Peak Detector tab Figure 20 You may need to optimize these settings In house validation should be performed Notes 1 Select full range or partial range for the analysis range When using a partial range choose an appropriate analysis range based on your data Choose a start point after the primer peak and just before the rst de ned internal lane standard peak to help ensure proper sizing of the internal lane standard 2 The peak amplitude thresholds are the minimum peak heights at which the software will call a peak
56. e not intended as a comprehensive guide for using GeneMapper ID X software We recommend that users contact Applied Biosystems for training on the software 1 Select Tools then GeneMapper ID X Manager 2 Select the Analysis Methods tab 3 Select New and a new analysis method dialog box will open 4 In the Analysis Method Editor window select GeneMapper ID X Security Group as the Security Group This allows access for all users of the software Other security groups may be used 5 Enter a descriptive name for the analysis method such as PowerPlexY23 6 Select the Allele tab Figure 13 7 Select the bins text le that was imported in Section 6 A 10018TA Figure 13 The GeneMapper ID X Software Version 1 2 Allele tab 32 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD035 Revised 3 15 www promega com 6 D Creating a Casework Analysis Method with GeneMapper ID X Software Version 1 2 continued 8 Ensure that the Use marker speci c stutter ratio and distance if available box is checked Doing this will assign locus speci c stutter lters and distances from the imported stutter le We recommend the settings shown in Figure 13 for proper ltering of stutter peaks when using the PowerPlex Y23 System Notes 1 The GeneMapper ID X stutter les i
57. e punch during pretreatment may help alleviate static problems 10 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD035 Revised 3 15 www promega com 4 B Direct Ampli cation of DNA from Storage Card Punches continued Ampli cation Setup 1 Thaw the PowerPlex Y23 5X Master Mix PowerPlex Y23 10X Primer Pair Mix and Water Ampli cation Grade completely Note Centrifuge tubes brie y to bring contents to the bottom then vortex reagents for 15 seconds before each use Do not centrifuge the 10X Primer Pair Mix or 5X Master Mix after vortexing as this may cause the reagents 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 ampli cation mix for all samples It also ensures that each reaction contains the same PCR ampli cation mix 3 Use a clean MicroAmp plate for reaction assembly and label appropriately 4 Add the nal volume of each reagent listed in Table 2 to a sterile tube Table 2 PCR Ampli cation Mix for Direct Ampli cation of DNA from Storage Card Punches PCR Ampli cation Mix Component1 Volume Per Reaction
58. eMapper ID software version 3 2 complete the Applied Biosystems GeneMapper ID Software Human Identi cation 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 For analysis using GeneMapper ID software version 3 2 you will need the proper panels and bins text les PowerPlexY23_Panels_vX x txt and PowerPlexY23_Bins_vX x txt les where X x refers to the most recent version of the panels and bins text les Note Run les generated using the Applied Biosystems 3500 or 3500xL Genetic Analyzer cannot be analyzed using GeneMapper ID Software Version 3 2 You must analyze these les with GeneMapper ID X software version 1 2 or later Getting Started 1 To obtain the proper panels and bins text les and CC5_ILS_500 xml le for the PowerPlex Y23 System go to www promega com resources tools genemapper id software panels and bin sets 2 Select the PowerPlex System that you are using and select GeneMapper ID Enter your contact information and select Submit 3 Save the PowerPlexY23_Panels_vX x txt and PowerPlexY23_Bins_vX x txt les where X x refers to the most recent version of the panels and bins text les to a known location on your computer 4 Save the CC5_ILS_500 xml le to a known location on your computer Importing Panels and Bins Text File
59. ed by the User 95 C dry heating block water bath or thermal cycler crushed ice ice water bath or a freezer plate block centrifuge compatible with 96 well plates aerosol resistant pipette tips 3500 3500xL capillary array 36cm plate retainer and base set standard POP 4 polymer for the Applied Biosystems 3500 or 3500xL Genetic Analyzer anode bu er container cathode bu er container MicroAmp optical 96 well plate and septa or equivalent Hi Di formamide Applied Biosystems Cat 4311320 The quality of formamide is critical Use Hi Di formamide Freeze formamide in aliquots at 20 C Multiple freeze thaw cycles or long term storage at 4 C may cause breakdown of formamide Poor quality formamide 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 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 At the rst use thaw the CC5 Internal Lane Standard 500 Y23 and PowerPlex Y23 Allelic Ladder Mix completely After the rst use store the reagents at 2 10 C for up to 1 month Note Centrifuge tubes brie y to bring contents to the bottom t
60. ed ice or a freezer plate block or in an ice water bath immediately prior to capillary electrophoresis Do not cool samples in a thermal cycler set at 4 C as this may lead to artifacts due to DNA re annealing Poor capillary electrophoresis injection CC5 ILS 500 Y23 peaks also a ected Re inject the sample Check the laser power Poor quality formamide was used Use only Hi Di formamide when analyzing samples Faint or absent allele peaks Improper storage of the 2800M Control DNA for the positive control reaction Extra peaks visible in one Contamination with another template DNA or previously or all color channels ampli ed 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 a freezer plate block or in an ice water bath immediately prior to capillary electrophoresis Do not cool samples in a thermal cycler set at 4 C as this may lead to artifacts due to DNA re annealing Double stranded DNA migrates faster than single stranded DNA during capillary electrophoresis Appearance of shadow peaks migrating in front of the main peaks especially if the shadow peaks are separated by the same distance as the main peaks in a heterozygote can indicate the presence of double stranded DNA due to incomplete denaturation or post injection re annealing Artifacts
61. ed 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 28 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD035 Revised 3 15 www promega com 5 B Detection of Ampli ed Fragments Using POP 4 Polymer and the ABI PRISM 3100 or 3100 Avant Genetic Analyzer with Data Collection Software Version 2 0 or the Applied Biosystems 3130 or 3130xl Genetic Analyzer with Data Collection Software Version 3 0 continued 6 In the spectral viewer select dye set G5 and con rm that the active dye set is the le generated for the PowerPlex 5 dye chemistry It is critical to select the correct G5 spectral for the PowerPlex 5 dye chemistry If the PowerPlex 5 dye chemistry is not the active dye set locate the PowerPlex 5 dye spectral in the List of Calibrations for Dye Set G5 and select Set 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 c
62. esholds are usually 50 150RFU for data generated on the ABI PRISM 3100 and 3100 Avant Genetic Analyzers and Applied Biosystems 3130 and 3130xl Genetic Analyzers For the Applied Biosystems 3500 and 3500xL Genetic Analyzers Life Technologies suggests an analysis threshold of 175RFU under their default injection conditions However individual laboratories should determine their peak amplitude thresholds from internal validation studies Peak heights for the CC5 ILS 500 Y23 are generally lower than those for the other dyes Therefore the threshold for the orange dye may be lower than that for the other dyes 10 Select the Peak Quality tab You may change the settings for peak quality Note For Steps 10 and 11 see the GeneMapper ID X user s manual for more information The settings in Steps 10 and 11 should be based on the results of your internal validation 11 Select the SQ amp GQ Settings tab You may change these settings 36 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD035 Revised 3 15 www promega com 6 E Creating a Databasing or Paternity Analysis Method Using a Global Filter with GeneMapper ID X Software Version 1 2 continued 12 Select Save to save the new analysis method 13 Select Done to exit the GeneMapper ID X Manager Processing Data for Databasing or Paternity Samples 1 Select
63. genotyping Note The positive control DNA de ned in the GeneMapper ID X panel le is the 2800M Control DNA Rede ne the genotype in the panel le if using a di erent positive control DNA 5 In the Analysis Method column select the analysis method created previously in this section 6 In the Panel column select the panels text le that was imported in Section 6 A 7 In the Size Standard column select the size standard that was imported in Section 6 B or created in Section 6 C 8 Select Analyze green arrow button to start data analysis Note By default the software is set to display the Analysis Requirements Summary window and Allelic Ladder Analysis Summary window if an issue is detected After analysis is complete the default setting is to show the Analysis Summary tab If these default settings are changed manual troubleshooting may be necessary 34 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD035 Revised 3 15 www promega com 6 D Creating a Casework Analysis Method with GeneMapper ID X Software Version 1 2 continued 9 If all analysis requirements are met the Save Project window will open Figure 15 Figure 15 The Save Project window 10 Enter the project name 11 Choose the applicable security group from the drop down menu and then select OK When the analysis is
64. hen vortex for 15 seconds before each use Do not centrifuge after vortexing as this may cause the reagents to be concentrated at the bottom of the tube 2 Prepare a loading cocktail by combining and mixing CC5 ILS 500 Y23 and Hi Di formamide as follows 1 0 l CC5 ILS 500 Y23 samples 10 l Hi Di formamide samples Be sure to use the CC5 ILS 500 Y23 as the size standard when using the PowerPlex Y23 System Do not use the CC5 ILS 500 Cat DG1521 The CC5_ILS_500 xml le can be used to assign fragment sizes for the CC5 ILS 500 Y23 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 based on laboratory preferences Keep the volume of formamide at 10 0 l per well and adjust the volume added to the wells in Step 4 accordingly 16 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD035 Revised 3 15 www promega com 5 A Detection of Ampli ed Fragments Using the Applied Biosystems 3500 or 3500xL Genetic Analyzer with 3500 Data Collection Software Version 1 0 continued 3 Vortex for 10 15 seconds to mix 4 Pipet 11 l of formamide internal lane standard mix into each well 5 Add 1 l of ampli ed sample or 1 l of PowerPlex Y23 Allelic Ladder Mix to each well Cover wells with
65. iate precautions when handling this substance Always wear gloves and safety glasses when working with formamide Sample Preparation 1 At the rst use thaw the CC5 Internal Lane Standard 500 Y23 and PowerPlex Y23 Allelic Ladder Mix After the rst use store the reagents at 2 10 C for up to 1 month Note Centrifuge tube brie y to bring contents to the bottom then vortex for 15 seconds before each use Do not centrifuge after vortexing as this may cause the reagents to be concentrated at the bottom of the tube 2 Prepare a loading cocktail by combining and mixing CC5 ILS 500 Y23 and Hi Di formamide as follows 1 0 l CC5 ILS 500 Y23 samples 10 0 l Hi Di formamide samples Be sure to use the CC5 ILS 500 Y23 as the size standard when using the PowerPlex Y23 System Do not use the CC5 ILS 500 Cat DG1521 The CC5_ILS_500 xml le can be used to assign fragment sizes for the CC5 ILS 500 Y23 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 based on laboratory preferences Keep the volume of formamide at 10 0 l per well and adjust the volume added to the wells in Step 4 accordingly 3 Vortex for 10 15 seconds to mix 4 Pipet 11 l of formamide internal lane standard mix into each well Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA
66. ical reaction plates with punches from the same samples 4 Amplify samples using the thermal cycling protocol provided above but subject each plate to a di erent cycle number For initial testing amplify using the following cycle numbers Additional testing may be required Blood sample on one 1 2mm FTA or pretreated nonFTA punch 25 26 and 27 cycles Buccal cells on two 1 2mm FTA punches 26 27 and 28 cycles Buccal cells on one 1 2mm FTA punch 27 28 and 29 cycles Buccal cells on one 1 2mm pretreated nonFTA punch 25 26 and 27 cycles 5 Following ampli cation use your laboratory s validated separation and detection protocols to determine the optimal cycle number for the sample type and number of storage card punches 4 C Direct Ampli cation of DNA from Swabs Materials to Be Supplied by the User GeneAmp PCR System 9700 with a gold plated or silver plated sample block Applied Biosystems centrifuge compatible with a 96 well plate or reaction tubes MicroAmp optical 96 well reaction plate or 0 2ml MicroAmp reaction tubes Applied Biosystems aerosol resistant pipette tips SwabSolution Kit Cat DC8271 Pretreat OmniSwab GE Healthcare or cotton swabs with the SwabSolution Kit Cat DC8271 as described in the SwabSolution Kit Technical Manual TMD037 to generate a swab extract Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free i
67. ict product liability or otherwise 2015 Promega Corpora on All Rights Reserved Plexor and PowerPlex are registered trademarks of Promega Corpora on AmpSolu on DNA IQ Iden ty Automa on PunchSolu on and SwabSolu on are trademarks of Promega Corpora on ABI PRISM Applied Biosystems GeneAmp GeneMapper and MicroAmp are registered trademarks of Applied Biosystems Bode Buccal DNA Collector is a trademark of the Bode Technology Group Inc EasiCollect and OmniSwab are trademarks of Whatman FTA is a registered trademark of Flinders Technologies Pty Ltd and is licensed to Whatman Hi Di is a trademark of Applera Corpora on POP 4 is a registered trademark of Life Technologies Corpora on POP 6 and POP 7 are trademarks of Life Technologies Corpora on Sampact is a trademark of Fitzco Vacutainer is a registered trademark of Becton Dickinson and Company Products may be covered by pending or issued patents or may have certain limita ons Please visit our Web site for more informa on All prices and speci ca ons are subject to change without prior no ce Product claims are subject to change Please contact Promega Technical Services or access the Promega online catalog for the most up to date informa on on Promega products
68. inciples and Applications for DNA Ampli cation 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 Gusm o L and Carracedo A 2003 Y chromosome speci c STRs Pro les in DNA 6 1 3 6 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 65 www promega com TMD035 Revised 3 15 11 Jobling M A Pandya A and Tyler Smith C 1997 The Y chromosome in forensic analysis and paternity testing Int J Legal Med 110 118 24 12 Gill P et al 2001 DNA Commission of the International Society of Forensic Genetics Recommendations on forensic analysis using Y chromosome STRs Int J Legal Med 114 305 9 13 Roewer L et al 2001 Online reference database of European Y chromosomal short tandem repeat STR haplotypes Forensic Sci Int 118 106 13 14 Butler J M et al 2002 A novel multiplex for simultaneous ampli cation of 20 Y chromosome STR markers Forensic Sci Int 129 10 24 15 Kayser M et al 1997 Evaluation of Y chromosomal STRs A multicenter study Int J Legal Med 110 125 33 16 Ruitberg C M Reeder D J and Butler J M 2001 STRBase A short tandem repeat DNA databa
69. jection time is 23 seconds and the injection voltage is 1 2kV Change the run time to 1 500 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 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 G5 in the dye set drop down list Select OK 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 en
70. li cation reaction volume Insu cient template Use the recommended amount of template DNA if available High salt concentration or altered pH If the DNA template is stored in TE bu er 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 a ect PCR A change in pH also may a ect PCR Store DNA in TE 4 bu er 10mM Tris HCl pH 8 0 0 1mM EDTA TE 4 bu er with 20 g ml glycogen or nuclease free water Faint or absent peaks may be seen more often when using the maximum template volume or reduced ampli cation reaction volume The reaction volume was too low This system is optimized for a nal reaction volume of 25 l Decreasing the reaction volume may result in suboptimal performance Extra peaks visible in one Artifacts of STR ampli cation Ampli cation of excess amounts or all color channels of puri ed DNA can result in a higher number of artifact peaks Use the recommended amount of template DNA See Section 6 L for additional information about stutter and artifacts Ampli cation of excess amounts also may result in overampli cation and signal saturation If signal is saturated repeat ampli cation with less swab extract or reduced cycle number Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356
71. lick the plate graphic that corresponds to the plate on the autosampler that contains your ampli ed samples 9 When the plate record is linked to the plate the plate graphic changes from yellow to green and the green Run Instrument arrow will become 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 40 minutes 6 Data Analysis 6 A Importing PowerPlex Y23 Panels Bins and Stutter Text Files into GeneMapper ID X Software Version 1 2 The instructions in this section were written using GeneMapper ID X software version 1 2 Due to potential di erences between individual software versions some of the instructions may not apply to other software versions To facilitate analysis of data generated with the PowerPlex Y23 System we have created panels and bins text les to allow automatic assignment of genotypes using GeneMapper ID X software We recommend that users receive training from Applied Biosystems on the GeneMapper ID X software to familiarize themselves with proper operation of the software Notes 1 The panels bins and stutter text les mentioned here are compatible with earlier versions of the GeneMapper ID X software 2 The GeneMapper ID X stutter les include lters for the plus st
72. m the drop down menu Figure 9 9254TA Figure 9 De ning plate properties 5 Select Assign Plate Contents Figure 10 9255TA Figure 10 Assigning plate contents Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 25 www promega com TMD035 Revised 3 15 6 Assign sample names to wells 7 In the lower left portion of the screen under Assays use the Add from Library option to select the Assay created in Step 2 d or one previously created Click on the Add to Plate button and close the window 8 Under File Name Conventions use the Add from Library option to select the File Name Convention created in Step 2 e or one previously created Click on the Add to Plate button and close the window 9 Under Results Groups use the Add from Library option to select the Results Group created in Step 2 f or one previously created Click on the Add to Plate button and close the window 10 Highlight the sample wells and then select the boxes in the Assays File Name Conventions and Results Groups that pertain to those samples 11 Select Link Plate for Run 12 The Load Plate window will appear Select Yes 13 In the Run Information window Figure 11 assign a Run Name Select Start Run not shown 9256TA Figure 11 Assigning a run name 26 Promega Corpora on 2800 Woods H
73. minute extension step at 60 C after thermal cycling Section 4 Decrease cycle number Increase the nal extension time Peak height imbalance Excessive amount of DNA Ampli cation of gt 20ng of template can result in an imbalance with smaller loci showing more product than larger loci Be sure to use the recommended number of punches Follow the manufacturer s recommendations when depositing sample onto the card Decrease cycle number The reaction volume was too low This system is optimized for a nal reaction volume of 25 l to overcome inhibitors present in FTA cards and PunchSolution Reagent Decreasing the reaction volume may result in suboptimal performance The cycle number was too high Decrease the cycle number by one cycle and repeat the ampli cation Ampli cation was inhibited when using more than one storage card punch with blood Use only one 1 2mm storage card punch with blood DNA was not accessible on nonlytic material Small loci may amplify preferentially with large loci dropping out Pretreat nonFTA materials with PunchSolution Reagent to ensure that DNA is liberated from cellular proteins 56 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD035 Revised 3 15 www promega com 7 C Direct Ampli cation of DNA From Storage Card Punches continued Symptoms Causes an
74. mith J R et al 1995 Approach to genotyping errors caused by nontemplated nucleotide addition by Taq DNA polymerase Genome Res 5 312 7 35 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 36 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 37 Mandrekar P V Krenke B E and Tereba A 2001 DNA IQ The intelligent way to purify DNA Pro les in DNA 4 3 16 38 Krenke B E et al 2005 Development of a novel uorescent two primer approach to quantitative PCR Pro les in DNA 8 1 3 5 9 Appendix 9 A Advantages of Using the Loci in the PowerPlex Y23 System The loci included in the PowerPlex Y23 System Tables 6 and 7 were selected because they represent well characterized loci generally accepted for forensic use This multiplex includes all loci in the European minimal haplotype DYS19 DYS385a b DYS389I II DYS390 DYS391 DYS392 and DYS393 see www yhrd org the Scienti c Working Group DNA Analysis Methods SWGDAM recommended Y STR panel European minimal haplotype plus DYS438 and DYS439 and the loci included in the US Y STR database SWGDAM recommended loci plus DYS437 DYS456 DYS458 DYS635 DYS448 and Y GATA H4 Six
75. n 3 2 These instructions are intended as a guide to start analyzing data in GeneMapper ID software They are not intended as a comprehensive guide for using GeneMapper ID software We recommend that users contact Applied Biosystems for training on the software These instructions loosely follow the Applied Biosystems GeneMapper ID software tutorial pages 5 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 40 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD035 Revised 3 15 www promega com 6 I Creating a Casework Analysis Method with GeneMapper ID Software Version 3 2 continued 5 Enter a descriptive name for the analysis method such as PowerPlexY23 6 Select the Allele tab Figure 19 7 Select the bins text le that was imported in Section 6 F 8 Ensure that the Use marker speci c stutter ratio if available box is checked 9 Enter the values shown in Figure 19 for proper ltering of stutter peaks when using the PowerPlex Y23 System For an explanation of the proper usage and e ects of these settings refer to th
76. n USA 800 356 9526 608 274 4330 Fax 608 277 2516 13 www promega com TMD035 Revised 3 15 Ampli cation Setup 1 Thaw the PowerPlex Y23 5X Master Mix PowerPlex Y23 10X Primer Pair Mix and Water Ampli cation Grade completely Note Centrifuge tubes brie y to bring contents to the bottom then vortex reagents for 15 seconds before each use Do not centrifuge the 10X Primer Pair Mix or 5X Master Mix after vortexing as this may cause the reagents 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 ampli cation mix for all samples It also ensures that each reaction contains the same PCR ampli cation mix 3 Use a clean MicroAmp plate for reaction assembly and label appropriately Alternatively determine the number of clean 0 2ml reaction tubes required and label appropriately 4 Add the nal volume of each reagent listed in Table 3 to a sterile tube Table 3 PCR Ampli cation Mix for Direct Ampli cation of DNA from Swabs PCR Ampli cation Mix Component1 Volume Per Reaction Number of Reactions Final Volume Water Ampli cation Grade 15 5 l PowerPlex Y23 5X Master Mix 5 0 l Po
77. nclude lters for the plus stutter associated with the two trinucleotide repeat loci DYS481 and DYS392 as well as lters for the plus 2 and minus 2 base artifacts associated with the DYS19 locus 2 If you do not check the Use marker speci c stutter ratio and distance if available box you will need to optimize these settings In house validation should be performed 9 Select the Peak Detector tab Figure 14 You may need to optimize these settings In house validation should be performed 8259TA Figure 14 The GeneMapper ID X Software Version 1 2 Peak Detector tab Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 33 www promega com TMD035 Revised 3 15 Notes 1 Select full range or partial range for the analysis range When using a partial range choose an appropriate analysis range based on your data Choose a start point after the primer peak and just before the rst de ned internal lane standard peak to help ensure proper sizing of the internal lane standard 2 The peak amplitude thresholds are the minimum peak heights at which the software will call a peak Values for peak amplitude thresholds are usually 50 150RFU for data generated on the ABI PRISM 3100 and 3100 Avant Genetic Analyzers and Applied Biosystems 3130 and 3130xl Genetic Analyzers For the Applied Biosystems 3500 and 3500xL Genetic Analy
78. ncubation DNA was not accessible on nonlytic material Pretreat swabs with SwabSolution Reagent to ensure that DNA is liberated from cellular proteins Faint or absent peaks for the If the positive control reaction failed to amplify check to make positive control reaction sure that the correct amount of 2800M Control DNA was added to the reaction Due to the reduced cycle numbers used with swab extracts it is necessary to increase the mass of 2800M Control DNA to obtain a pro le We recommend 5ng of 2800M Control DNA per 25 l ampli cation reaction This mass of DNA should be reduced if cycle number is increased and decreased if the cycle number is increased Increase or decrease by twofold the mass of 2800M Control DNA for every one cycle decrease or increase respectively Improper storage of the 2800M Control DNA Extra peaks visible in one or Swab extract was contaminated Assemble a reaction containing all color channels the swab extract prepared from a blank swab or assemble a reaction where the SwabSolution Reagent is processed and incubated as a blank without a swab Artifacts of STR ampli cation Ampli cation of swab extracts with high DNA concentrations can result in artifact peaks due to overampli cation resulting in saturated signal on the CE instrument We recommend 2 l of swab extract per reaction Using more than 2 l in a 25 l reaction or using 2 l with a smaller reaction volume may result in o
79. ns Final Volume Water Ampli cation Grade to a nal volume of 25 0 l PowerPlex Y23 5X Master Mix 5 0 l PowerPlex Y23 10X Primer Pair Mix 2 5 l template DNA 0 5ng 2 3 4 up to 17 5 l total reaction volume 25 l 1Add Water Ampli cation Grade to the tube rst then add PowerPlex Y23 5X Master Mix and PowerPlex Y23 10X Primer Pair Mix The template DNA will be added at Step 6 2Store DNA templates in TE 4 bu er 10mM Tris HCl pH 8 0 0 1mM EDTA or TE 4 bu er with 20 g ml glycogen If the DNA template is stored in TE bu er that is not pH 8 0 or contains a higher EDTA concentration the volume of DNA added should not exceed 20 of the nal reaction volume PCR ampli cation e ciency 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 di er depending on the DNA quanti cation method used 22 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 based on your DNA quanti cation method 4The PowerPlex Y23 System was optimized and balanced for 0
80. ollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD035 Revised 3 15 www promega com 5 B Detection of Ampli ed Fragments Using POP 4 Polymer and the ABI PRISM 3100 or 3100 Avant Genetic Analyzer with Data Collection Software Version 2 0 or 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 ice water bath or freezer plate block centrifuge compatible with 96 well plates aerosol resistant pipette tips 3100 or 3130 capillary array 36cm plate retainer and base set standard POP 4 polymer for the 3130 3130xl Genetic Analyzers 10X genetic analyzer bu er with EDTA MicroAmp optical 96 well plate and septa or equivalent Hi Di formamide Applied Biosystems Cat 4311320 The quality of formamide is critical Use Hi Di formamide Freeze formamide in aliquots at 20 C Multiple freeze thaw cycles or long term storage at 4 C may cause breakdown of formamide Poor quality formamide 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 Formamide is an irritant and a teratogen avoid inhalation and contact with skin Read the warning label and take appropr
81. olymer pouch and perform a spatial calibration Samples may be analyzed as described in the Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 1 Open the 3500 Data Collection Software The Dashboard screen will launch Figure 2 To ensure that you are viewing the most up to date information press the Refresh button Ensure that the Consumables Information and Maintenance Noti cations are acceptable Set the oven temperature to 60 C then select Start Pre Heat When the Oven Temperature and Detection Cell Temperature turn green you may proceed with the rst injection 9247TA Figure 2 The Dashboard 2 Prior to the rst analysis using the PowerPlex Y23 System you must create an Instrument Protocol Size Standard QC Protocol Assay File Name Convention and Results Group a To create a new Instrument Protocol navigate to the Library select Instrument Protocols and then select Create Alternatively a previously created Instrument Protocol may be used Figure 3 shows the settings used at Promega for the Applied Biosystems 3500xL Genetic Analyzer for the application type dye set capillary length polymer run module and appropriate protocol information The only setting that was changed from the default settings is dye set 18 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD035 Revised 3 15
82. ontinued Run was too short and larger peaks in ILS were not captured Not all CC5 ILS 500 Y23 peaks de ned 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 A low quality allelic ladder was used during analysis Ensure that only high quality allelic ladders are used for analysis O ladder alleles An allelic ladder from a di erent run than the samples was used Re analyze samples with an allelic ladder from the same run The GeneMapper ID X 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 D or 6 E Panels text le selected for analysis was incorrect for the STR system used Assign correct panels text le that corresponds to the STR system used for ampli cation The allelic ladder was not identi ed as an allelic ladder in the Sample Type column The internal lane standard was not properly identi ed in the sample Manually rede ne the sizes of the size standard fragments in the sample A low quality allelic ladder was used during analysis Ensure that only high quality allelic ladders are used for analysis Size standard not called correctly Starting data point was incorrect for the partial range
83. ools then GeneMapper Manager 2 Select the Size Standard tab 3 Select Import 4 Browse to the location of the CC5_ILS_500 xml le on your computer 5 Highlight the le and then select Import 6 Select Done to save changes and close the GeneMapper Manager 6 H Creating a Size Standard with GeneMapper ID Software Version 3 2 1 Select Tools then GeneMapper Manager 2 Select the Size Standard tab 3 Select New 4 Select Basic or Advanced Figure 17 The type of analysis method selected must match the type of analysis method created earlier Select OK 5725TA Figure 17 The Select Dye and Analysis Method window Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 39 www promega com TMD035 Revised 3 15 5 Enter a detailed name such as CC5 ILS 500 Y23 advanced in the Size Standard Editor Figure 18 8199TA Figure 18 The GeneMapper ID Software Version 3 2 Size Standard Editor 6 Choose Orange for the Size Standard Dye 7 Enter the sizes of the internal lane standard fragments 60 65 80 100 120 140 160 180 200 225 250 275 300 325 350 375 400 425 450 475 and 500 bases See Section 9 D Figure 24 8 Select OK 6 I Creating a Casework Analysis Method with GeneMapper ID Software Versio
84. r 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 CC5 ILS 500 Y23 peaks de ned 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 amplitude threshold in the analysis method for the orange channel to include peaks or increase the volume of CC5 ILS 500 Y23 used in Section 5 If peaks are low quality rede ne the size standard for the sample to skip these peaks An incorrect size standard was used Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 63 www promega com TMD035 Revised 3 15 Symptoms Causes and Comments 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 les are or analysis method is invalid set to the same mode either Classic or Basic or Advanced mode No alleles called but no error Panels text le was not selected for sample In the Panel column message appears select the appropriate panels text le for the STR system that was used No si
85. r instrument preparation in Section 5 The wrong spectral calibration was used Make sure that the spectral calibration was performed using the same polymer type as that for sample analysis i e do not use a POP 4 generated spectral calibration for a POP 7 run Error message after attempting to There was a con ict between di erent sets of panels and bins text import panels and bins text les les Check to be sure that the bins are installed properly If not Unable to save panel data delete all panels and bins text les and re import les in a java SQLEException ORA 00001 di erent order unique constraint IFA CKP_NNN violated 64 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD035 Revised 3 15 www promega com 7 F GeneMapper ID Software continued Symptoms Causes and Comments Allelic ladder peaks GeneMapper ID software was not used or microsatellite labeled o 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 di erences using the allelic ladder We recommend 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 veri ed b
86. rd with GeneMapper ID X Software Version 1 2 30 6 D Creating a Casework Analysis Method with GeneMapper ID X Software Version 1 2 31 6 E Creating a Databasing or Paternity Analysis Method Using a Global Filter with GeneMapper ID X Software Version 1 2 34 6 F Importing PowerPlex Y23 Panels and Bins Text Files with GeneMapper ID Software Version 3 2 37 6 G Importing the CC5 ILS 500 Size Standard into GeneMapper ID Software Version 3 2 38 6 H Creating a Size Standard with GeneMapper ID Software Version 3 2 38 6 I Creating a Casework Analysis Method with GeneMapper ID Software Version 3 2 39 6 J Creating a Databasing or Paternity Analysis Method Using a Global Filter with GeneMapper ID Software Version 3 2 42 6 L Results 45 2 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD035 Revised 3 15 www promega com 1 Description STR
87. re incubated for 30 minutes or until wells are dry Incubation for shorter time periods may result in incomplete inactivation of the PunchSolution Reagent We have not tested longer incubation times Inactive PunchSolution Reagent was used to pretreat nonFTA punches Thaw the PunchSolution Reagent at 2 10 C Do not store reagents in the refrigerator door where the temperature can uctuate Do not refreeze as this may reduce activity Faint or absent allele peaks Positive control did not amplify Check to make sure that the for the positive control reaction correct amount of 2800M Control DNA was added to the reaction We recommend 5ng of 2800M Control DNA per 25 l ampli cation reaction Do not include a blank punch in the positive control reaction Presence of a blank punch may inhibit ampli cation of 2800M Control DNA Optimize the amount of 2800M Control DNA for your thermal cycling conditions and laboratory preferences Improper storage of the 2800M Control DNA Extra peaks visible in one Punch was contaminated Take punches from blank paper or all color channels between samples Ampli cation of processed punches with high amounts of DNA can result in artifact peaks due to overampli cation resulting in saturating signal on the CE instrument Be sure to use the recommended number of punches Use of a larger punch size or a smaller reaction volume may result in overampli cation and signal satura
88. ributor Contact information available at www promega com E mail genetic promega com 7 A Ampli cation and Fragment Detection This section provides information about general ampli cation and detection For questions about ampli cation of extracted DNA see Section 7 B For questions about direct ampli cation see Sections 7 C and 7 D Symptoms Causes and Comments Faint or absent allele peaks The PowerPlex Y23 5X Master Mix was not vortexed well before use Vortex the 5X Master Mix for 15 seconds before dispensing into the PCR ampli cation mix An air bubble formed at the bottom of the reaction tube Use a pipette to remove the air bubble or centrifuge the reactions brie y before thermal cycling Thermal cycler plate or tube problems Review the thermal cycling protocol in Section 4 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 Vortex the PowerPlex Y23 10X Primer Pair Mix for 15 seconds before use Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 49 www promega com TMD035 Revised 3 15 Symptoms Causes and Comments Faint or absent allele peaks continued Samples were not denatured completely Heat denature samples for the recommended time then cool on crush
89. rifuge the plate or tubes to bring contents to the bottom of the wells and remove any air bubbles 14 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD035 Revised 3 15 www promega com 4 C Direct Ampli cation of DNA from Swabs continued Thermal Cycling Ampli cation and detection instrumentation may vary You will need to optimize protocols including the amount of template DNA cycle number injection conditions and loading volume for your laboratory instrumentation Testing at Promega shows that 26 cycles works well for a variety of sample types Cycle number will need to be optimized in each laboratory for each sample type that is ampli ed see below 1 Place the MicroAmp plate or reaction tubes in the thermal cycler 2 Select and run the recommended protocol which is provided below Be sure to select Max mode as the ramp speed This requires a silver plated or gold plated sample block The ramp speed is set after the thermal cycling run is started When the Select Method Options screen appears select Max for the ramp speed and enter the reaction volume The estimated total cycle time is approximately 1 5 hours Thermal Cycling Protocol 96 C for 2 minutes then 94 C for 10 seconds 61 C for 1 minute 72 C for 30 seconds for 26 cycles then 60 C for 20 minutes 4 C soak 3 After completion of
90. rimer peak and just before the rst de ned internal lane standard peak to help ensure proper sizing of the internal lane standard 2 The peak amplitude thresholds are the minimum peak heights at which the software will call a peak Values for peak amplitude thresholds are usually 50 150RFU for data generated on the ABI PRISM 3100 and 3100 Avant Genetic Analyzers and Applied Biosystems 3130 and 3130xl Genetic Analyzers However individual laboratories should determine their peak amplitude thresholds from internal validation studies Peak heights for the CC5 ILS 500 Y23 are generally lower than those for the other dyes Therefore the threshold for the orange dye may be lower than that for the other dyes 11 Select the Peak Quality tab You may change the settings for peak quality Note See the GeneMapper ID user s manual for more information The settings in Steps 11 and 12 should be based on the results of your internal validation 12 Select the Quality Flags tab You may change these settings 13 Select OK to save your settings 44 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD035 Revised 3 15 www promega com 6 J Creating a Databasing or Paternity Analysis Method Using a Global Filter with GeneMapper ID Software Version 3 2 continued Processing Data for Databasing or Paternity Samples 1 Select
91. rpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 45 www promega com TMD035 Revised 3 15 6 L Results Representative results of the PowerPlex Y23 System are shown in Figure 22 The PowerPlex Y23 Allelic Ladder Mix is shown in Figure 23 10718TA A B C D E Figure 22 The PowerPlex Y23 System The 2800M Control DNA 0 5ng was ampli ed using the PowerPlex Y23 System Ampli cation products were mixed with CC5 Internal Lane Standard 500 Y23 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 uorescein labeled loci DYS576 DYS389I DYS448 DYS389II and DYS19 Panel B An electropherogram showing the peaks of the JOE labeled loci DYS391 DYS481 DYS549 DYS533 DYS438 and DYS437 Panel C An electropherogram showing the peaks of the TMR ET labeled loci DYS570 DYS635 DYS390 DYS439 DYS392 and DYS643 Panel D An electropherogram showing the peaks of the CXR ET labeled loci DYS393 DYS458 DYS385a b DYS456 and Y GATA H4 Panel E An electropherogram showing the 60bp to 425bp fragments of the CC5 Internal Lane Standard 500 Y23 46 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 27
92. ry window was not active and there was an analysis requirement that was not met Turn on Analysis Requirement Summary in the Options menu and correct the necessary analysis requirements to continue analysis Edits in label edit viewer cannot be viewed To view edits made to a project the project rst must be saved Close the plot view window return to the main GeneMapper ID X page and save the project Display the plot window again and then view the label edit table Marker header bar for some loci are gray When an edit is made to a locus the quality ags and marker header bar automatically change to gray To change the GQ and marker header bar for a locus to green override the GQ in the plot window Alleles not called To analyze samples with GeneMapper ID X software at least one allelic ladder must be de ned An insu cient number of CC5 ILS 500 Y23 fragments was de ned Be sure to de ne at least two CC5 ILS 500 Y23 fragments smaller than the smallest sample peak and at least two CC5 ILS 500 Y23 fragments larger than the largest sample peak In this instance the allelic ladder would have failed the allelic ladder quality check 60 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD035 Revised 3 15 www promega com 7 E GeneMapper ID X Software continued Symptoms Causes and Comments Alleles not called c
93. s 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 Panel Manager icon in the upper left navigation pane 4 Select File then Import Panels 5 Navigate to the panels text le downloaded in the Getting Started section Select the le then Import 6 In the navigation pane highlight the PowerPlex Y23 panels folder that you just imported in Step 5 7 Select File then Import Bin Set 8 Navigate to the bins text le downloaded in the Getting Started section Select the le then Import 9 At the bottom of the Panel Manager window select OK This will save the panels and bins text les and close the window 38 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD035 Revised 3 15 www promega com 6 G Importing the CC5 ILS 500 Size Standard into GeneMapper ID Software Version 3 2 There are two options when creating a size standard Use this protocol or the alternative protocol in Section 6 H The CC5_ILS_500 xml le can be used to assign fragment sizes for the CC5 ILS 500 Y23 and can be downloaded at www promega com resources tools genemapper id software panels and bin sets 1 Select T
94. s select Max for the ramp speed and enter the reaction volume The estimated total cycle time is 1 5 hours Thermal Cycling Protocol 96 C for 2 minutes then 94 C for 10 seconds 61 C for 1 minute 72 C for 30 seconds for 26 cycles then 60 C for 20 minutes 4 C soak 3 After completion of the thermal cycling protocol proceed with fragment analysis or store ampli ed samples at 20 C in a light protected box Note Long term storage of ampli ed samples at 4 C or higher may produce artifacts 12 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD035 Revised 3 15 www promega com 4 B Direct Ampli cation of DNA from Storage Card Punches continued PCR Optimization Cycle number should be optimized based on the results of an initial experiment to determine the sensitivity with your collection method sample types number of punches and instrumentation 1 Choose several samples that represent typical sample types you encounter in the laboratory Prepare them as you would using your normal work ow 2 Depending on your preferred protocol place one or two 1 2mm storage card punches containing buccal cells or one 1 2mm punch of a storage card containing whole blood in each well of a reaction plate Be sure to pretreat nonFTA samples with the PunchSolution Kit Cat DC9271 3 Prepare three ident
95. se for the human identity testing community Nucleic Acids Res 29 320 2 17 Prinz M et al 1997 Multiplexing of Y chromosome speci c STRs and performance for mixed samples Forensic Sci Int 85 209 18 18 Prinz M et al 2001 Validation and casework application of a Y chromosome speci c STR multiplex Forensic Sci Int 120 177 88 19 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 Identi cation 1992 Promega Corporation 245 69 20 Hartmann J M et al 1991 Guidelines for a quality assurance program for DNA analysis Crime Laboratory Digest 18 44 75 21 Internal Validation Guide of Y STR Systems for Forensic Laboratories GE713 Promega Corporation 22 Kline M C et al 2005 Results from the NIST 2004 DNA quantitation study J Forensic Sci 50 570 8 23 Levinson G and Gutman G A 1987 Slipped strand mispairing A major mechanism for DNA sequence evolution Mol Biol Evol 4 203 21 24 Schl tterer C and Tautz D 1992 Slippage synthesis of simple sequence DNA Nucleic Acids Res 20 211 5 25 Lim S K et al 2007 Variation of 52 new Y STR loci in the Y chromosome consortium worldwide panel of 76 diverse individuals Int J Legal Med 121 124 7 26 Kayser M et al 2004 A comprehensive survey of human Y chromosomal microsatelli
96. se marker speci c stutter ratio and distance if available box is checked Doing this will assign locus speci c stutter lters and distances from the imported stutter le Ensure that the appropriate global lter is applied to this analysis method For example for a 20 lter enter 0 20 for the Global Cut o Value for Tri Tetra and Penta repeats Figure 16 Note If you do not check the Use marker speci c stutter ratio and distance if available box you will need to optimize these settings In house validation should be performed Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 35 www promega com TMD035 Revised 3 15 10019TA Figure 16 The GeneMapper ID X Software Version 1 2 Allele tab with settings for using a 20 peak lter 9 Select the Peak Detector tab Figure 14 You may need to optimize these settings In house validation should be performed Notes 1 Select full range or partial range for the analysis range When using a partial range choose an appropriate analysis range based on your data Choose a start point after the primer peak and just before the rst de ned internal lane standard peak to help ensure proper sizing of the internal lane standard 2 The peak amplitude thresholds are the minimum peak heights at which the software will call a peak Values for peak amplitude thr
97. ser Guide to edit a library entry Assign a descriptive protocol name Note For more detailed information refer to the Applied Biosystems 3500 3500xL Genetic Analyzers User Guide b To create a new Size Standard for the QC protocol navigate to the Library Select Size Standards and then select Create Alternatively a previously created Size Standard may be used Assign the size standard the name ILS500 or another appropriate name Choose Orange as the Dye Color The fragments in the size standard are 60 65 80 100 120 140 160 180 200 225 250 275 300 325 350 375 400 425 450 475 and 500 bases See Figure 4 9227TA Figure 4 The Create New Size Standard window 20 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD035 Revised 3 15 www promega com 5 A Detection of Ampli ed Fragments Using the Applied Biosystems 3500 or 3500xL Genetic Analyzer with 3500 Data Collection Software Version 1 0 continued c To create a new QC Protocol navigate to the Library Select QC Protocols and then select Create Alternatively a previously created QC Protocol may be used Assign a descriptive protocol name such as CC5 ILS 500 Y23 Select the size standard created in Step 2 b The settings for the QC protocol should be based on the internally validated conditions for the PowerPlex Y23 Sys
98. tem on the Applied Biosystems 3500 or 3500xL Genetic Analyzer Figure 5 shows one option for these settings Note Peak heights for the CC5 ILS 500 Y23 are generally lower than those for the other dyes Therefore the threshold for the orange dye may be lower than that for the other dyes 9228TA Figure 5 The Create New QC Protocol window Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 21 www promega com TMD035 Revised 3 15 d To create a new Assay navigate to the Library Select Assays and then select Create Alternatively a previously created Assay may be used In the Create New Assay window Figure 6 select the Instrument Protocol created in Step 2 a and the QC Protocol created in Step 2 c Assign a descriptive assay name Select the application type HID An Assay is required for all named samples on a plate Note If autoanalysis of sample data is desired refer to the instrument user s manual for instructions 9229TA Figure 6 The Create New Assay window 22 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD035 Revised 3 15 www promega com 5 A Detection of Ampli ed Fragments Using the Applied Biosystems 3500 or 3500xL Genetic Analyzer with 3500 Data Collection Software Version 1 0 continued e
99. ter 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 select dye set G5 and con rm that the active dye set is the le generated for POP 7 polymer and PowerPlex 5 dye chemistry It is critical to select the correct G5 spectral for the PowerPlex 5 dye chemistry and that the G5 spectral was generated using POP 7 polymer If the PowerPlex 5 dye chemistry is not the active dye set locate the POP 7 PowerPlex 5 dye spectral in the List of Calibrations for Dye Set G5 and select Set 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 graphic that corresponds to the plate on the autosampler that contains your ampli ed samples Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 73 www promega com TMD035 Revised 3 15 9 When the plate record is linked to the plate the plate graphic changes 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
100. tes Am J Hum Genet 74 1183 97 27 Vermeulen M et al 2009 Improving global and regional resolution of male lineage di erentiation by simple single copy Y chromosomal short tandem repeat polymorphisms Forensic Sci Int Genet 3 205 13 28 Rodig H et al 2008 Evaluation of haplotype discrimination capacity of 35 Y chromosomal short tandem repeat loci Forensic Sci Int 174 182 8 29 Geppert M Edelmann J and Lessig R 2009 The Y chromosomal STRs DYS481 DYS570 DYS576 and DYS643 Leg Med Tokyo 11 S109 10 66 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD035 Revised 3 15 www promega com 8 References continued 30 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 31 Gill P et al 1997 Considerations from the European DNA Pro ling Group EDNAP concerning STR nomenclature Forensic Sci Int 87 185 92 32 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 33 Budowle B et al 2008 Null allele sequence structure at the DYS448 locus and implications for pro le interpretation Int J Legal Med 122 421 7 34 S
101. the Applied Biosystems 3500 or 3500xL Genetic Analyzer Repeat sample preparation using fresh formamide Long term storage of ampli ed sample in formamide can result in artifacts 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 Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 51 www promega com TMD035 Revised 3 15 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 di erent injection of allelic ladder to determine sizes Poor injection of allelic ladder Include more than one ladder per instrument run Internal size standard was not assigned correctly Evaluate the sizing labels on the CC5 ILS 500 Y23 and correct if necessary Peak height imbalance Miscellaneous balance problems At the rst use thaw the 10
102. the navigation pane highlight the PowerPlex Y23 panels folder that you just imported in Step 5 7 Select File then Import Bin Set 8 Navigate to the bins text le downloaded in the Getting Started section Select the le then Import 9 In the navigation pane highlight the PowerPlex Y23 panels folder that you just imported in Step 5 10 Select File then Import Marker Stutter A warning box will appear asking if you want to overwrite current values Select Yes 11 Navigate to the stutter text le imported in the Getting Started section Select the le then Import 12 At the bottom of the Panel Manager window select OK This will save the panels bins and stutter text les and close the window 6 B Importing the CC5 ILS 500 IDX Size Standard into GeneMapper ID X Software Version 1 2 There are two options when creating a size standard Use this protocol or the alternative protocol in Section 6 C The CC5_ILS_500_IDX xml le can be used to assign fragment sizes for the CC5 ILS 500 Y23 and can be downloaded at www promega com resources tools genemapper id software panels and bin sets 1 Select Tools then GeneMapper ID X Manager 2 Select the Size Standard tab 3 Select Import 4 Navigate to the location of the CC5_ILS_500_IDX xml le on your computer 5 Highlight the le and then select Import 6 Select
103. the thermal cycling protocol proceed with fragment analysis or store ampli ed samples at 20 C in a light protected box Note Long term storage of ampli ed samples at 4 C or higher may produce artifacts PCR Optimization Cycle number should be optimized based on the results of an initial experiment to determine the sensitivity with your collection method sample types and instrumentation 1 Choose several samples that represent typical sample types you encounter in the laboratory Prepare them as you would using your normal work ow 2 Prepare three identical reaction plates with aliquots of the same swab extracts 3 Amplify samples using the thermal cycling protocol provided above but subject each plate to a di erent cycle number 25 26 and 27 cycles Note This recommendation is for 2 l of swab extract Additional cycle number testing may be required 4 Following ampli cation use your laboratory s validated separation and detection protocols to determine the optimal cycle number for the sample type Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 15 www promega com TMD035 Revised 3 15 5 Instrument Setup and Sample Preparation 5 A Detection of Ampli ed Fragments Using the Applied Biosystems 3500 or 3500xL Genetic Analyzer with 3500 Data Collection Software Version 1 0 Materials to Be Suppli
104. tion Reagent to ensure that DNA is liberated from cellular proteins Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 59 www promega com TMD035 Revised 3 15 Symptoms Causes and Comments Extreme variability in sample There can be signi cant individual to individual variability in to sample peak heights cell deposition onto buccal swabs This will appear as variability in peak heights between swab extracts The extraction process maximizes recovery of ampli able DNA from buccal swabs but does not normalize the amount of DNA present If variability is extreme quantify the DNA using a uorescence based double stranded DNA quanti cation method or qPCR based quanti ca tion method The quanti cation values can be used to normalize input template amounts to minimize variation in signal intensity 7 E GeneMapper ID X Software Symptoms Causes and Comments Stutter peaks not ltered Stutter text le was not imported into the Panel Manager when the panels and bins text les were imported Be sure that the Use marker speci c stutter ratio and distance if available box is checked If the Use marker speci c stutter ratio and distance if available box is not checked stutter distance must be de ned in the Analysis Method Allele tab Samples in the project not analyzed The Analysis Requirement Summa
105. tion If the signal is saturated repeat the ampli ca tion with a smaller punch a larger reaction volume or reduced cycle number Ampli cation of excess template for a given cycle number can result in overloading of the capillary upon electrokinetic injection The presence of excess DNA in the capillary makes it di cult to maintain the DNA in a denatured single stranded state Some single stranded DNA renatures and becomes double stranded Double stranded DNA migrates faster than single stranded DNA during capillary electrophoresis and appears as shadow peaks migrating in front of the main peaks i e smaller in size Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 55 www promega com TMD035 Revised 3 15 Symptoms Causes and Comments Extra peaks visible in one Artifacts of STR ampli cation Direct ampli cation of gt 20ng of or all color channels continued template can result in a higher number of artifact peaks Use the recommended punch size and number of punches Do not reduce the reaction volume below 25 l Optimize the cycle number See Section 6 L for additional information about stutter and artifacts Artifacts of STR ampli cation Ampli cation of STRs 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 a 20
106. tion 5 The wrong spectral calibration was used Make sure that the spectral calibration was performed using the same polymer type as that for sample analysis e g do not use a POP 4 generated spectral calibration for a POP 7 run 7 F GeneMapper ID 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 di erent an error is obtained To analyze samples with GeneMapper ID software at least one allelic ladder must be de ned An insu cient number of CC5 ILS 500 Y23 fragments was de ned Be sure to de ne at least two CC5 ILS 500 Y23 fragments smaller than the smallest sample peak or allelic ladder peak and at least two CC5 ILS 500 Y23 fragments larger than the largest sample peak or allelic ladder peak Run was too short and larger peaks in ILS were not captured Not all CC5 ILS 500 Y23 peaks de ned 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 A low quality allelic ladder was used during analysis Ensure that only high quality allelic ladders are used for analysis 62 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 F
107. ts pending b 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 Gesellscha zur F rderung der Wissenscha en e V Germany c TMR ET CXR ET and CC5 dyes are proprietary d This product or por ons thereof is manufactured and sold under license from GE Healthcare under Australia Pat No 692230 Austria Pat No E236994 Belgium Pat No 0743987 Canada Pat No 2231475 EP Pat Nos 0743987 and 0851867 France Pat Nos 0743987 and 0851867 Germany Pat Nos 19581489 69530286 8 and 0851867 Italy Pat Nos 0743987 and 0851867 Japan Pat No 3066984 Liechtenstein Pat Nos 0743987 and 0851867 Netherlands Pat Nos 0743987 and 0851867 Spain Pat Nos 2197193 and 2173310 Sweden Pat Nos 0743987 and 0851867 Switzerland Pat Nos 0743987 and 0851867 United Kingdom Pat Nos 0743987 and 0851867 U S Pat Nos 5 654 419 5 688 648 5 869 255 6 177 247 5 707 804 6 028 190 6 544 744 7 015 000 and 5 728 528 and other pending and foreign patent applica ons End User Terms and Condi ons Acceptance These terms and condi ons shall govern the purchase use transfer and acceptance of the products described in the purchase order quota on or invoice which products are sold and distributed by Promega to the buyer transferee of such products the End User The transfer sale of products to the End User is expressly condi onal upon End User s acceptance of these terms
108. utter associated with the two trinucleotide repeat loci DYS481 and DYS392 as well as lters for plus 2 and minus 2 base artifacts associated with the DYS19 locus Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 29 www promega com TMD035 Revised 3 15 Getting Started 1 To obtain the proper panels bins and stutter text les and CC5_ILS_500_IDX xml le for the PowerPlex Y23 System go to www promega com resources tools genemapper id software panels and bin sets 2 Select the PowerPlex System that you are using and select GeneMapper ID X Enter your contact information and select Submit 3 Save the PowerPlexY23_Panels_IDX_vX x txt PowerPlexY23_Bins_IDX_vX x txt and PowerPlexY23_Stutter_ IDX_vX x txt les where X x refers to the most recent version of the panels bins and stutter text les to a known location on your computer 4 Save the CC5_ILS_500_IDX xml le to a known location on your computer Importing Panels Bins and Stutter Text Files 1 Open the GeneMapper ID X software 2 Select Tools then Panel Manager 3 Highlight the Panel Manager icon in the upper left navigation pane 4 Select File then Import Panels 5 Navigate to the panels text le downloaded in the Getting Started section Select the le then Import 6 In
109. verampli cation and signal saturation If signal is saturated repeat ampli cation with less swab extract or reduced cycle number Ampli cation of excess template for a given cycle number resulted in overloading of the capillary upon electrokinetic injection Excess DNA in the capillary is di cult to maintain in a denatured single stranded state Some single stranded DNA renatures and becomes double stranded Double stranded DNA migrates faster than single stranded DNA during capillary electrophoresis and appears as shadow peaks migrating in front of the main peaks i e smaller in size 58 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD035 Revised 3 15 www promega com 7 D Direct Ampli cation of DNA From Swabs continued Symptoms Causes and Comments Extra peaks visible in one or Artifacts of STR ampli cation Ampli cation of STRs can result all color channels continued 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 a 20 minute extension step at 60 C after thermal cycling Section 4 Use 2 l of swab extract in a PowerPlex Y23 reaction A larger volume of swab extract may contain more than the recommended amount of DNA template resulting in incomplete adenylation Decrease cycle number Increase the
110. viewer window in the data collection software Each injection will take approximately 45 minutes 9 C DNA Extraction and Quanti cation Methods and Automation Support Promega o ers a wide variety of reagents and automated methods for sample preparation DNA puri cation and DNA quanti cation prior to STR ampli cation For analysis of database reference and other single source samples we recommend direct ampli cation of DNA from FTA card punches or direct ampli cation of DNA from swabs and nonFTA punches following a preprocessing step with the SwabSolution Kit or PunchSolution Kit respectively The SwabSolution Kit Cat DC8271 contains reagents for rapid DNA preparation from buccal swabs prior to ampli cation The procedure lyses cells contained on the swab head and releases into solution su cient DNA for STR ampli cation A small volume of the nal swab extract is added to the PowerPlex reaction The PunchSolution Kit is used to process punches from nonFTA storage cards containing blood or buccal samples prior to direct ampli cation For casework or samples that require DNA puri cation we recommend the DNA IQ System Cat DC6700 which is a DNA isolation system designed speci cally for forensic samples 37 This system uses paramagnetic particles to prepare clean samples for STR analysis easily and e ciently and can be used to extract DNA from stains or liquid samples such as blood
111. werPlex Y23 10X Primer Pair Mix 2 5 l swab extract 2 0 l total reaction volume 25 l 1Add Water Ampli cation Grade to the tube rst and then add PowerPlex Y23 5X Master Mix and PowerPlex Y23 10X Primer Pair Mix The swab extract will be added at Step 6 5 Vortex the PCR ampli cation mix for 5 10 seconds and then pipet 23 l of PCR ampli cation mix into each reaction well or tube Failure to vortex the PCR ampli cation mix su ciently can result in poor ampli cation or locus to locus imbalance 6 Pipet 2 0 l of swab extract for each sample into the appropriate well of the reaction plate or tube 7 For the positive ampli cation control vortex the tube of 2800M DNA dilute an aliquot to 2 5ng l and add 2 l to a reaction well or tube containing 23 l of PCR ampli cation mix Note Optimization of the amount of 2800M Control DNA may be required depending on thermal cycling conditions and laboratory preferences 8 For the negative ampli cation control pipet Water Ampli cation Grade or TE 4 bu er instead of swab extract into a reaction well containing PCR ampli cation mix Note Additional negative controls can be included Assemble a reaction containing the swab extract prepared from a blank swab or assemble a reaction where the SwabSolution Reagent is processed as a blank without a swab 9 Seal or cap the plate or close the tubes Optional Brie y cent
112. www promega com 5 A Detection of Ampli ed Fragments Using the Applied Biosystems 3500 or 3500xL Genetic Analyzer with 3500 Data Collection Software Version 1 0 continued 9393TA Figure 3 The Create New Instrument Protocol window The recommended settings are Application Type HID Capillary Length 36cm Polymer POP 4 Dye Set G5 Promega G5 spectral Run Module HID36_POP4 xl Injection Time1 15 seconds for the Applied Biosystems 3500 Genetic Analyzer 24 seconds for the Applied Biosystems 3500xL Genetic Analyzer Injection Voltage 1 2kV Run Time 1 210 1 500 seconds 1Injection time may be modi ed to increase or decrease peak heights When creating an Instrument Protocol be sure to select the same dye set that was used to perform the Promega 5 dye spectral calibration We recommend using a run time of 1 210 1 500 seconds and the default injection conditions Run time and other instrument settings should be optimized and validated in your laboratory Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 19 www promega com TMD035 Revised 3 15 When optimizing injection conditions in your laboratory you may choose to create speci c Instrument Protocols for each condition tested If a single Instrument Protocol is used follow the instructions in the Applied Biosystems 3500 3500xL Genetic Analyzers U
113. x Y23 5X Master Mix PowerPlex Y23 10X Primer Pair Mix 2800M Control DNA and Water Ampli cation Grade are provided in a separate box and should be stored separately from those used following ampli cation PowerPlex Y23 Allelic Ladder Mix and CC5 Internal Lane Standard 500 Y23 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 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 3 B Spectral Calibration Proper spectral calibration is critical to evaluate multicolor systems with the ABI PRISM 3100 and 3100 Avant Genetic Analyzers and Applied Biosystems 3130 3130xl 3500 and 3500xL Genetic Analyzers Spectral calibration must be performed for each individual instrument For protocols and additional information about spectral calibration on these instruments see the PowerPlex 5 Dye Matrix Standards 3100 3130 Technical Bulletin TBD024 This manual is available online at www promega com protocols 6 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 433
114. y opening the analysis method using the GeneMapper Manager and then selecting the General tab The analysis type cannot be changed If the method is not HID delete it and create a new analysis method Contact Promega Technical Services at genetic promega com with questions 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 Identi cation 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 ve 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 ampli cation of DNA by 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 Pr
115. ze standard was selected In the Size Standard column be sure to select the appropriate size standard Size standard was not correctly de ned or size peaks were missing Rede ne 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 agged as red and no allele sizes will be called Error message The bins text le assigned to the analysis method was deleted In Both the Bin Set used in the Analysis the GeneMapper Manager select the Analysis Methods tab and Method and the Panel must belong open the analysis method of interest Select the Allele tab and to the same Chemistry Kit select an appropriate bins text le The wrong bins text le was chosen in the analysis method Allele tab Be sure to choose the appropriate bins text le as shown in Figure 19 Signi cantly raised baseline Poor spectral calibration Perform a new spectral calibration and re run the samples 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 method Advanced mode analysis methods and size standards are recommended Incorrect G5 spectral was active Re run samples and con rm that the PowerPlex 5 dye G5 spectral is set for G5 See instructions fo
116. ze that some laboratories use POP 7 polymer and therefore have included a protocol in this manual Some DNA independent artifacts migrate in the panel range with the POP 7 polymer see Table 9 Global lters used for database analysis will generally lter these artifact peaks However these peaks may be labeled with casework samples Internal validation should be performed and interpretation guidelines created that describe the artifacts and their impact on data analysis For information about DNA dependent stutter products and artifacts see Table 4 in Section 6 L Table 9 DNA Independent Artifacts Dye Label Instrument Artifact Size Fluorescein Applied Biosystems 3130 Genetic Analyzers with POP 7 polymer 65 68 bases 73 75 bases 85 87 bases 100 104 bases1 JOE Applied Biosystems 3130 Genetic Analyzers with POP 7 polymer 66 69 bases 88 91 bases1 1The signal strength of these artifacts increases with storage of the ampli cation plate at 4 C sometimes in as short a time period as overnight but more commonly when plates are left at 4 C for a few days We recommend storing ampli cation products at 20 C Note For data analysis follow the instructions in Section 6 except use POP 7 speci c panels and bins text les e g use PowerPlexY23_POP7_Panels_IDX_vX x txt instead of PowerPlexY23_Panels_IDX_vX x txt Contact Promega Technical Services at genetic promega com for the
117. zers Life Technologies suggests an analysis threshold of 175RFU under their default injection conditions However individual laboratories should determine their peak amplitude thresholds from internal validation studies Peak heights for the CC5 ILS 500 Y23 are generally lower than those for the other dyes Therefore the threshold for the orange dye may be lower than that for the other dyes 10 Select the Peak Quality tab You may change the settings for peak quality Note For Steps 10 and 11 see the GeneMapper ID X user s manual for more information The settings in Steps 10 and 11 should be based on the results of your internal validation 11 Select the SQ amp GQ Settings tab You may change these settings 12 Select Save to save the new analysis method 13 Select Done to exit the GeneMapper ID X Manager Processing Data for Casework Samples 1 Select File then New Project 2 Select Edit then Add Samples to Project 3 Browse to the location of the run les Highlight desired les and then select Add to list followed by Add 4 In the Sample Type column use the drop down menu to select Allelic Ladder Sample Positive Control or Negative Control as appropriate for the sample Every folder in the project must contain at least one allelic ladder injection that is designated as Allelic Ladder in the Sample Type column for proper

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PowerPlex® Y23 System