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PowerPlex® 16 HS Technical Manual

1. 34 6 A PowerPlex 16 Panels Bins and Stutter Text Files with GeneMapper ID X Software Version 1 2 34 6 B Creating a Size Standard with GeneMapper ID X Software Version 1 2 35 6 C Creating a Casework Analysis Method with GeneMapper ID X Software Version 1 2 36 6 D Creating a Databasing or Paternity Analysis Method with GeneMapper ID X Software Version 1 2 39 6 E PowerPlex Panels and Bins Text Files with GeneMapper ID Software Version 3 2 42 6 F Creating a Size Standard with GeneMapper ID Software Version 3 2 43 6 G Creating a Casework Analysis Method with GeneMapper ID Software Version 3 2 45 6 H Creating a Databasing or Paternity Analysis Method with GeneMapper ID Software Version 3 2 48 6 I Controls 50 6 J Results 51 7 Troubleshooting
2. 81 9 G Related Products 82 10 Summary of Changes 83 Promega Corporation 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 TMD022 Revised 9 15 The PowerPlex 16 HS System provides all materials necessary to amplify STR regions of human genomic DNA including hot start Taq DNA polymerase which is a component of the PowerPlex HS 5X Master Mix This manual contains protocols for use of the PowerPlex 16 HS System with GeneAmp PCR system 9600 and 9700 thermal cyclers in addition to protocols to separate amplified products and detect separated material Figure 1 Protocols to operate the fluorescence detection instruments should be obtained from the instrument manufacturer Information on other Promega fluorescent STR systems including the PowerPlex 16 Monoplex Systems is available upon request from Promega or online at www promega com Amplification Setup Thermal Cycling Instrument Setup and Sample Preparation Data Analysis Figure 1 An overview of the PowerPlex 16 HS System protocol Section 4 Section 4 Section 5 Sections 6
3. 6 4 A Amplification of Extracted DNA 7 4 B Direct Amplification of DNA from Storage Card Punches 10 4 C Direct Amplification of DNA from Swabs 14 5 Instrument Setup and Sample Preparation 17 5 A Detection of Amplified Fragments Using the Applied Biosystems 3500 or 3500xL Genetic Analyzer 17 5 B Detection of Amplified Fragments Using 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 27 5 C Detection of Amplified Fragments Using the ABI PRISM 3100 Genetic Analyzer with Data Collection Software Version 1 0 1 or 1 1 30 5 D Detection of Amplified Fragments Using the ABI PRISM 310 Genetic Analyzer 32 6 Data Analysis
4. 53 7 A Amplification and Fragment Detection 53 7 B Direct Amplification from Storage Card Punches 56 7 C Amplification of DNA from Swabs 58 7 D GeneMapper ID X Software 60 2 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD022 Revised 9 15 www promega com 1 Description STR short tandem repeat loci consist of short repetitive sequence elements 3 7 base pairs in length 1 4 These repeats are well distributed throughout the human genome and are a rich source of highly polymorphic markers which may be detected using the polymerase chain reaction 5 9 Alleles of STR loci are differentiated by the number of copies of the repeat sequence contained within the amplified region and are distinguished from one another using fluorescence detection following electrophoretic separation The PowerPlex 16 HS System a c is used for human identification applications including forensic analysis relationship testing and research use The system a
5. then GeneMapper ID X Manager 2 Select the Size Standard tab 3 Select New 4 The Size Standard Editor box will open Figure 13 Enter a detailed name such as ILS 600 5 Choose a security group from the drop down menu 6 Choose Red for the Size Standard Dye 7 Enter the sizes of the internal lane standard fragments 60 80 100 120 140 160 180 200 225 250 275 300 325 350 375 400 425 450 475 500 550 and 600 bases See Section 9 D Figure 28 Note Definition and detection of the 600bp fragment is optional 8 Select OK 9429TA Figure 13 The GeneMapper ID X Size Standard Editor 36 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD022 Revised 9 15 www promega com 6 C 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 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 enter a descriptive name for the analysis me
6. 1 Select File then New Project 2 Select Edit then Add Samples to Project 3 Browse to the location of the run files Highlight desired files 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 5 In the Analysis Method column select the analysis method created previously in this section 6 In the Panel column select the panels text file that was imported in Section 6 E 7 In the Size Standard column select the size standard that was created in Section 6 F 8 If analyzing data from an ABI PRISM 310 Genetic Analyzer ensure that the appropriate matrix file is selected in the Matrix column 9 Select Analyze green arrow button to start the data analysis 6 I Controls 1 Observe the results for the negative control Using the protocols defined in this manual the negative control should be devoid of amplification products 2 Observe the results for the 2800M Control DNA Compare the 2800M Control DNA allelic repeat sizes with the locus specific allelic ladder The expected 2800M Control DNA allele designations for each locus are listed in Table 4 Se
7. 10 If all analysis requirements are met the Save Project window will open Figure 16 9430TA Figure 16 The Save Project window 11 Enter the project name 12 Choose the applicable security group from the drop down menu then select OK When the analysis is finished 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 Navigate to the Genotype tab or Samples tab To assist the review of any low quality samples use the default Data Interpretation plot settings and review the contents in the Quality Value Details table The values displayed in the Analysis Method Peak Quality and SQ amp GQ Settings tabs are defaults and will affect the quality values displayed in the plot settings We recommend that you modify the values in these tabs to fit your laboratory s data analysis protocols 6 D Creating a Databasing or Paternity 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 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 ana
8. 3 Use a clean MicroAmp plate for reaction assembly and label appropriately Promega Corporation 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 TMD022 Revised 9 15 4 Add the final volume of each reagent listed in Table 3 to a sterile tube Table 3 PCR Amplification Mix for Direct Amplification of DNA From Swabs PCR Amplification Mix Component1 Volume Per Reaction Number of Reactions Final Volume Water Amplification Grade 12 5 l PowerPlex HS 5X Master Mix 5 0 l PowerPlex 16 HS 10X Primer Pair Mix 2 5 l 5X AmpSolution Reagent 5 0 l swab extract 2 0 l total reaction volume 25 l 1Add Water Amplification Grade to the tube first then add PowerPlex HS 5X Master Mix and PowerPlex 16 HS 10X Primer Pair Mix The swab extract will be added at Step 6 5 Vortex the PCR amplification mix for 5 10 seconds then pipet 23 l of PCR amplification mix into each reaction well Failure to vortex the PCR amplification mix sufficiently can result in poor amplification or locus to locus imbalance 6 Pipet 2 0 l of swab extract for each sample into the appropriate well of the reaction plate 7 For the positive amplification control vortex the tube of 2800M Control DNA then dilute an aliquot to 2 5ng l and add 2 l to a reaction well containing 23 l of PCR am
9. Fax 608 277 2516 TMD022 Revised 9 15 www promega com Table 2 The PowerPlex 16 HS System Locus Specific Information STR Locus Label Chromosomal Location GenBank Locus and Locus Definition Repeat Sequence1 5 3 Penta E FL 15q NA AAAGA D18S51 FL 18q21 3 HUMUT574 AGAA 22 D21S11 FL 21q11 21q21 HUMD21LOC TCTA Complex 22 TH01 FL 11p15 5 HUMTH01 human tyrosine hydroxylase gene AATG 22 D3S1358 FL 3p NA TCTA Complex FGA TMR ET 4q28 HUMFIBRA human fibrinogen alpha chain gene TTTC Complex 22 TPOX TMR ET 2p24 2pter HUMTPOX human thyroid peroxidase gene AATG D8S1179 TMR ET 8q24 13 NA TCTA Complex 22 vWA TMR ET 12p13 31 HUMVWFA31 human von Willebrand factor gene TCTA Complex 22 Amelogenin2 TMR ET Xp22 1 22 3 and Y HUMAMEL human Y chromosomal gene for Amelogenin like protein NA Penta D JOE 21q NA AAAGA CSF1PO JOE 5q33 3 34 HUMCSF1PO human c fms proto oncogene for CSF 1 receptor gene AGAT D16S539 JOE 16q24 1 NA GATA D7S820 JOE 7q11 21 22 NA GATA D13S317 JOE 13q22 q31 NA TATC D5S818 JOE 5q23 3 32 NA AGAT 1The August 1997 report 23 24 of the DNA Commission of the International Society for Forensic Haemogenetics ISFH states 1 for STR loci within coding genes the coding strand shall be used and the repeat sequence motif defined using the first possible 5 nucleotide of a repeat motif and 2 for STR
10. Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 79 www promega com TMD022 Revised 9 15 10 Create the appropriate table by selecting the PowerTable Make Allele Table or Make CODIS Table macro The three available table formats are shown below The PowerTable option allows up to four alleles per sample file Additional information such as low peak signal or high peak signal also is included The Allele Table and CODIS Table options include only two alleles per locus If more than two alleles are present at a locus the smallest alleles identified are included The Allele Table format displays the categories loci in columns while the CODIS table format displays the categories in rows These tables can be customized to fit needs To save data in tables go to the Table drop down menu highlight Export to File and save the file with the desired name and location The saved file can be viewed and analyzed using Microsoft Excel 11 Save the analyzed data Go to the File menu and select Save as The PowerTyper Macro is a Genotyper file and can be overwritten if Save is used instead of Save as PowerTable Format Sample Info Sample Comment Category Peak 1 Peak 2 Peak 3 Peak 4 Overflow Low Signal Saturation Edited Label Edited Row Allele Table Format Sample Info Category Allele 1 Category A
11. If this occurs at a heterozygous locus it is possible to observe the presence of two shadow peaks that differ in size by approximately the same distance as the single stranded alleles Peak height imbalance Excess DNA in the amplification 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 effect Use less swab extract or reduce the cycle number 60 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD022 Revised 9 15 www promega com 7 C Amplification of DNA from Swabs continued Symptoms Possible Causes and Comments Peak height imbalance continued Active protease carried over from swab extracts into the amplification reaction Larger loci are most susceptible to protease carryover and will drop out before the smaller loci Ensure that the heat block is heating to 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 protease inactivation Do not use an incubator set at 70 C to incubate tubes or plates Heat transfer is inefficient and will result in poor performance Only use a heat block to maintain efficient heat transfer Inactive SwabSolution Reagent Thaw the SwabSolution Reagent complete
12. Incorrect spectral was active Re run samples and confirm that the PowerPlex 4 dye F spectral calibration is set for F See instructions for instrument preparation in Section 5 7 E GeneMapper ID Software Symptoms Possible Causes and Comments Alleles not called To analyze samples with GeneMapper ID software the analysis parameters and size standard must both have Basic or Advanced as the analysis type If they are different an error is obtained Figure 25 To analyze samples with GeneMapper ID software at least one allelic ladder must be defined An insufficient number of ILS 600 fragments was defined Be sure to define at least two ILS 600 fragments smaller than the smallest sample peak or allelic ladder peak and at least two ILS 600 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 ILS 600 peaks defined in the size standard were detected during the run Create a new size standard using the internal lane standard fragments present in the sample Re run samples using a longer run time 5685TA Figure 25 The error message that appears in the GeneMapper ID software when the analysis parameters and size standard have different analysis types Promega Corporation 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 TMD022 R
13. Macro command because the labeled peak could not be found Peak heights for one or more alleles in the allelic ladder sample file were below 150RFU The allelic ladder categories are defined as having a minimum peak height of 150RFU If peak heights of ladder alleles are below 150RFU the software will not be able to locate the allele peak Re run the allelic ladder using more sample or longer injection time to assure peak heights above 150RFU CE spikes in the allelic ladder sample were identified as alleles by the macro Use a different injection of allelic ladder 66 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD022 Revised 9 15 www promega com 7 F PowerTyper 16 Macro continued Symptoms Possible Causes and Comments Error message Could not complete the Run Macro command because the labeled peak could not be found continued TH01 9 3 and 10 alleles were not separated when using heavy smoothing in the GeneScan analysis parameters Use light smoothing in the GeneScan analysis parameters Allelic ladder data were not compatible with the PowerTyper file used Confirm that the PowerTyper Macro file matches the allelic ladder being used The base pair size of alleles in the allelic ladder are outside of the defined category range Be sure internal lane standard fragments are correctly sized Redef
14. PowerPlex 16 HS Allelic Ladder Mix 1 150 l Internal Lane Standard ILS 600 P R O D U C T S I Z E C AT PowerPlex 16 HS System 400 reactions DC2100 Not For Medical Diagnostic Use This system contains sufficient reagents for 400 reactions of 25 l each Includes Pre amplification Components Box Blue Label 4 500 l PowerPlex HS 5X Master Mix 4 250 l PowerPlex 16 HS 10X Primer Pair Mix 25 l 2800M Control DNA 10ng l 10 1 250 l Water Amplification Grade Post amplification Components Box Yellow Label 4 50 l PowerPlex 16 HS Allelic Ladder Mix 4 150 l Internal Lane Standard ILS 600 The PowerPlex 16 HS Allelic Ladder Mix is provided in a separate sealed bag for shipping This component should be moved to the post amplification box after opening PowerPlex HS 5X Master Mix and PowerPlex 16 HS 10X Primer Pair Mix are manufactured as a matched set for optimal performance Do not combine components from kits with different lot numbers printed on the boxes and Certificates of Analysis If lots are mixed locus to locus imbalance and variation in signal intensity may occur Storage Conditions 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 The PowerPlex 16 HS 10X Primer Pair Mix PowerPlex 16 HS Allelic Ladder Mix and Internal Lane Standard 600 are light
15. This mass of DNA should be reduced if the cycle number used 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 Extra peaks visible in one or all color channels Swab extract was contaminated 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 Artifacts of STR amplification Amplification of swab extracts with high concentrations of DNA can result in artifact peaks due to overamplification resulting in saturated signal on the CE instrument We recommend 2 l of swab extract per 25 l reaction Using more than 2 l in a 25 l reaction or using 2 l with a smaller reaction volume may result in overamplification and signal saturation If signal is saturated repeat the amplification with less swab extract or a reduced cycle number Amplification of excess template for a given cycle number resulted in overloading of the capillary upon electrokinetic injection In addition to signal saturation excess DNA in the capillary is difficult 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
16. www promega com TMD022 Revised 9 15 4 The analysis parameters can be saved in the Params folder in most installations this is located at C AppliedBio Shared Analysis Sizecaller Params 5 Apply the stored analysis parameters file to the samples 6 Assign a new size standard Select a sample file and highlight the arrow next to size standard Select define new Assign the size standard peaks as shown in Figure 28 in Section 9 D Store the size standard in the Size Standards folder at C AppliedBio Shared Analysis Sizecaller SizeStandards 7 Apply the size standard file to the samples then analyze the sample files See below for additional information on the use of the PowerTyper 16 Macro Release 2 0 and Genotyper software Notes 1 Peak heights outside the linear range of the instrument may generate artifact peaks due to instrument saturation i e overloading the sample Bleedthrough pull ups from one color to another may be observed Saturated signal also may appear as two peaks split peak 2 If peak heights are not within the linear range of detection of the instrument the ratio of stutter peaks to real allele peaks increases and allele designations become difficult to interpret The balance of peak heights also may appear less uniform 3 There can be variation between instruments regarding the relative fluorescence levels detected using the same sample Furthermore different instruments vary in
17. 2800M Control DNA is sufficient to provide a robust profile using the cycle numbers recommended here A one cycle reduction in cycle number will require a twofold increase in mass of DNA template to generate similar signal intensity Similarly a one cycle increase in cycle number will require a twofold reduction in the amount of 2800M Control DNA to avoid signal saturation 8 Reserve a well containing PCR amplification mix as a negative amplification 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 the plate and briefly centrifuge the plate to bring storage card punches to the bottom of the wells and remove any air bubbles Note Place the amplification plate in the thermal cycler and start the thermal cycling program as soon as the PowerPlex System PCR amplification mix is added to all wells Prolonged storage of assembled reactions prior to cycling may result in poor performance i e lower peak heights for large amplicons Thermal Cycling Amplification 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 your laboratory instrumentation Testing at Promega shows that 27 cycles works well for a variety of sample types Buccal samples may require more amplification cycles than blood samples Cycle number will
18. 30 seconds ramp 29 to 60 C for 30 seconds ramp 23 to 70 C for 45 seconds for 10 cycles then 90 C for 30 seconds ramp 60 seconds to 60 C hold for 30 seconds ramp 50 seconds to 70 C hold for 45 seconds for 22 cycles then ramp 100 to 94 C for 30 seconds ramp 29 to 60 C for 30 seconds ramp 23 to 70 C for 45 seconds for 22 cycles then 60 C for 20 minutes 60 C for 20 minutes 4 C soak 4 C soak 1When using the GeneAmp PCR System 9700 thermal cycler the ramp rates indicated in the cycling program must be set and the program must be run in 9600 ramp mode The ramp rates are set in the Ramp Rate Modification screen While viewing the cycling program navigate to the Ramp Rate Modification screen by selecting More then Modify On the Ramp Rate Modification screen the default rates for each step are 100 The rate under each hold step is the rate at which the temperature will change to that hold temperature Figure 2 shows the appropriate ramp rates for the GeneAmp PCR System 9700 thermal cycler The ramp mode is set after start has been selected for the thermal cycling run A Select Method Options screen appears Select 9600 ramp mode and enter the reaction volume 10 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD022 Revised 9 15 www promega com 4 A Amplification of Extracted DNA conti
19. 6 14 D13S317 JOE 176 208 7 15 D5S818 JOE 119 155 7 16 1The length of each allele in the allelic ladder has been confirmed by sequence analyses 2When using an internal lane standard such as the Internal Lane Standard 600 the calculated sizes of allelic ladder components may differ from those listed This occurs because different sequences in allelic ladder and ILS components may cause differences in migration The dye label also affects migration of alleles 3For a current list of microvariants see the Variant Allele Report published at the U S National Institute of Standards and Technology NIST web site at www cstl nist gov div831 strbase 4Amelogenin is not an STR but displays a 106 base X specific band and a 112 base Y specific band 72 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD022 Revised 9 15 www promega com Table 4 The PowerPlex 16 HS System Allele Determinations in Commonly Available Standard DNA Templates Standard DNA Templates1 STR Locus K5622 9947A 99483 2800M Penta E 5 14 12 13 11 11 7 14 D18S51 15 16 15 19 15 18 16 18 D21S11 29 30 31 30 30 29 30 29 31 2 TH01 9 3 9 3 8 9 3 6 9 3 6 9 3 D3S1358 16 16 14 15 15 17 17 18 FGA 21 24 23 24 24 26 20 23 TPOX 8 9 8 8 8 9 11 11 D8S1179 12 12 13 13 12 13 14 15
20. 9251TA Figure 7 The Create New Assay window 24 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD022 Revised 9 15 www promega com 5 A Detection of Amplified Fragments Using the Applied Biosystems 3500 or 3500xL Genetic Analyzer continued 6 To create a new File Name Convention Figure 8 navigate to the Library Select File Name Conventions then select Create Alternatively a previously created File Name Convention may be used Select the File Name Attributes according to laboratory practices and save with a descriptive name 9252TA Figure 8 The Create New File Name Convention window 7 To create a new Results Group Figure 9 navigate to the Library Select Results Group then select Create Alternatively a previously created Results Groups may be used Select the Results Group Attributes according to laboratory practices Save with a descriptive name Promega Corporation 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 TMD022 Revised 9 15 8 To create a New Plate navigate to the Library and from the Manage menu select Plates then Create 9 Assign a descriptive plate name Select the plate type HID from the drop down menu Figure 10 9253TA Figure 9 The Create N
21. 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 TMD022 Revised 9 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 buffers and polymer 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 3 Ensure that the Consumables Information and Maintenance Notifications are acceptable Set the oven temperature to 60 C then select Start Pre Heat at least 30 minutes prior to the first injection to preheat the oven 9247TA Figure 3 The Dashboard 20 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD022 Revised 9 15 www promega com 5 A Detection of Amplified Fragments Using the Applied Biosystems 3500 or 3500xL Genetic Analyzer continued 2 To create a new Instrument Protocol navigate to the Library select Instrument Protocol then select Create Alternatively a previously created Instrument Protocol may be used Figure 4 shows the settings used at Promega for the Applied Biosystems 3500xL Genetic Analyzer for the appli
22. JOE 100 reactions DC6651 PowerPlex 18D System 200 reactions DC1802 800 reactions DC1808 PowerPlex 21 System 200 reactions DC8902 PowerPlex ESX 16 System 100 reactions DC6711 400 reactions DC6710 PowerPlex ESX 17 System 100 reactions DC6721 400 reactions DC6720 PowerPlex ESI 16 System 100 reactions DC6771 400 reactions DC6770 PowerPlex ESI 17 Pro System 100 reactions DC7781 400 reactions DC7780 PowerPlex CS7 System 100 reactions DC6613 PowerPlex Y23 System 50 reactions DC2305 200 reactions DC2320 Not for Medical Diagnostic Use Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 83 www promega com TMD022 Revised 9 15 Accessory Components Product Size Cat PowerPlex Matrix Standards 310 50 l each dye DG4640 PowerPlex Matrix Standards 3100 3130 25 l each dye DG4650 Internal Lane Standard 600 150 l DG1071 Water Amplification Grade 5 1 250 l DW0991 2800M Control DNA 10ng l 25 l DD7101 2800M Control DNA 0 25ng l 500 l DD7251 PunchSolution Kit 100 preparations DC9271 SwabSolution Kit 100 preparations DC8271 5X AmpSolution Reagent 500 l DM1231 Not for Medical Diagnostic Use Sample Preparation and Quantification Systems Product Size Cat DNA IQ System 100 reactions DC6701 400
23. 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 file that was imported in the Getting Started Section above Select the file then Import 6 In the navigation pane highlight the PowerPlex 16 panels folder that you just imported in Step 5 7 Select File then Import Bin Set 8 Navigate to the bins text file that was imported in the Getting Started Section above Select the file then Import 9 At the bottom of the Panel Manager window select OK The Panel Manager window will close automatically 6 F 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 18 The type of analysis method selected must match the type of analysis method created earlier Select OK 44 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD022 Revised 9 15 www promega com 6 F Creating a Size Standard with GeneMapper ID Software Version 3 2 continued 5725TA Figure 18 The Select Dye and Analysis Method window 5 Enter a detailed name such as ILS 600 Advanced in the Size Standard Editor Figure 19 5
24. Promega Corporation All Rights Reserved Maxwell Plexor and PowerPlex are registered trademarks of Promega Corporation AmpSolution Differex DNA IQ Identity Automation PowerTyper PunchSolution Slicprep and SwabSolution are trademarks of Promega Corporation ABI PRISM Applied Biosystems GeneMapper GeneScan Genotyper and MicroAmp are registered trademarks of Applied Biosystems Bode Buccal DNA Collector is a trademark of the Bode Technology Group Inc Excel Microsoft Windows and Windows NT are registered trademarks of Microsoft Corporation FTA is a registered trademark of Flinders Technologies Pty Ltd and is licensed to Whatman GenBank is a registered trademark of the U S Dept of Health and Human Services GeneAmp is a registered trademark of Roche Molecular Systems Inc Hi Di is a trademark of Applera Corporation Macintosh is a registered trademark of Apple Computer Inc POP 4 is a registered trademark of Life Technologies Corporation Products may be covered by pending or issued patents or may have certain limitations Please visit our Web site for more information All prices and specifications are subject to change without prior notice Product claims are subject to change Please contact Promega Technical Services or access the Promega online catalog for the most up to date information on Promega products
25. The preferred protocol for use with the GeneAmp PCR System 9700 thermal cycler is provided below Thermal Cycling Protocol1 96 C for 2 minutes then ramp 100 to 94 C for 30 seconds ramp 29 to 60 C for 30 seconds ramp 23 to 70 C for 45 seconds for 10 cycles then ramp 100 to 90 C for 30 seconds ramp 29 to 60 C for 30 seconds ramp 23 to 70 C for 45 seconds for 18 cycles then 60 C for 30 minutes 4 C soak 1When using the GeneAmp PCR System 9700 thermal cycler the ramp rates indicated in the cycling program must be set and the program must be run in 9600 ramp mode The ramp rates are set in the Ramp Rate Modification screen While viewing the cycling program navigate to the Ramp Rate Modification screen by selecting More then Modify On the Ramp Rate Modification screen the default rates for each step are 100 The rate under each hold step is the rate at which the temperature will change to that hold temperature Figure 2 shows the appropriate ramp rates for the GeneAmp PCR System 9700 thermal cycler The ramp mode is set after start has been selected for the thermal cycling run A Select Method Options screen appears Select 9600 ramp mode and enter the reaction volume 3 After completion of the thermal cycling protocol store amplified samples at 20 C in a light protected box Note Long term storage of amplified samples at 4 C or higher may produce artifacts Promega Corpora
26. a different injection of allelic ladder to determine sizes Poor injection of allelic ladder Include more than one ladder per instrument run Peak height imbalance Excessive amount of DNA Amplification of gt 1ng of template can result in an imbalance with smaller loci showing more product than larger loci Use less template or reduce the number of cycles in the amplification program by 2 4 cycles 10 20 or 10 18 cycling to improve locus to locus balance Note Dilution of overamplified samples can result in dropout of larger loci Degraded DNA sample DNA template was degraded and larger loci show diminished yield Repurify template DNA Insufficient template DNA Use the recommended amount of template DNA Stochastic effects can occur when amplifying low amounts of template Miscellaneous balance problems Thaw the 10X Primer Pair Mix and 5X Master Mix completely and vortex for 15 seconds before use Do not centrifuge the 10X Primer Pair Mix after mixing Calibrate thermal cyclers and pipettes routinely Using a 59 C annealing temperature instead of 60 C has been shown to improve balance in some instances Impure DNA template Inhibitors that may be present in forensic samples can lead to allele dropout or imbalance Impure DNA template Include a proteinase K digestion prior to DNA purification PCR amplification mix prepared in Section 4 was not mixed well Vortex the PCR amplification mix for 5 10 seconds before dispens
27. amplification program by 2 4 cycles to achieve the desired signal intensity Promega Corporation 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 TMD022 Revised 9 15 5 Centrifuge plate briefly to remove air bubbles from the wells 6 Denature samples at 95 C for 3 minutes then immediately chill on crushed ice or in an ice water bath for 3 minutes Denature samples just prior to loading the instrument Instrument Preparation Refer to the ABI PRISM 3100 Genetic Analyzer User Guide for instructions on cleaning the blocks installing the capillary array performing a spatial calibration and adding polymer to the reserve syringe 1 Open the ABI PRISM 3100 Data Collection Software 2 Change the GeneScan36_POP4DefaultModule module run time to 1 800 seconds 3 Change the injection voltage to 3kV 4 Change the injection time to 11 seconds Note Instrument sensitivities can vary Injection time and voltage may be adjusted in the Module Manager A suggested range for the injection time is 3 22 seconds and for the injection voltage is 1 3kV 5 Save the module with a new name e g GeneScan36_POP4PowerPlex16_3kV_11secs_1800 Use this as the initial run module for all runs 6 Open a new plate record Name the plate and select GeneScan Select the plate size 96 well Select Finish 7 Complet
28. and 9 Applied Biosystems 3500 or 3500xL Genetic Analyzer Section 5 A GeneAmp PCR System 9700 GeneAmp PCR System 9600 Applied Biosystems 3130 or 3130xl Genetic Analyzer with Data Collection Software Version 3 0 Section 5 B ABI PRISM 3100 or 3100 Avant Genetic Analyzer with Data Collection Software Version 2 0 Section 5 B ABI PRISM 3100 Genetic Analyzer with Data Collection Software Version 1 0 1 or 1 1 Section 5 C ABI PRISM 310 Genetic Analyzer Section 5 D GeneMapper ID X Software Version 1 2 Section 6 GeneScan Software and Windows Operating Systems Section 9 C GeneMapper ID Software Version 3 2 Section 6 GeneScan Software and Macintosh Operating Systems Section 9 C 4 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD022 Revised 9 15 www promega com 2 Product Components and Storage Conditions P R O D U C T S I Z E C AT PowerPlex 16 HS System 100 reactions DC2101 Not For Medical Diagnostic Use This system contains sufficient reagents for 100 reactions of 25 l each Includes Pre amplification Components Box Blue Label 1 500 l PowerPlex HS 5X Master Mix 1 250 l PowerPlex 16 HS 10X Primer Pair Mix 25 l 2800M Control DNA 10ng l 5 1 250 l Water Amplification Grade Post amplification Components Box Yellow Label 1 50 l
29. appropriate precautions when handling this substance Always wear gloves and safety glasses when working with formamide 28 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD022 Revised 9 15 www promega com 5 B Detection of Amplified Fragments Using 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 Sample Preparation 1 Thaw the Internal Lane Standard 600 Note Centrifuge tubes briefly to bring contents to the bottom then vortex for 15 seconds before each use Do not centrifuge after vortexing as this may cause the size standard to be concentrated at the bottom of the tube 2 Prepare a loading cocktail by combining and mixing Internal Lane Standard 600 and Hi Di formamide as follows 0 5 l ILS 600 samples 9 5 l Hi Di formamide samples Note The volume of internal lane standard used in the loading cocktail can be increased or decreased to adjust the intensity of the size standard peaks The optimal peak height for the 100 base fragment of the internal lane standard is 500 1 000RFU If peak heights are too low we recommend altering the formamide internal lane standard mix to contain 1 0 l of ILS 600 and 9 0 l of Hi Di formamide
30. at the bottom of the tube PowerPlex HS 5X Master Mix and PowerPlex 16 HS 10X Primer Pair Mix are manufactured as a matched set for optimal performance Do not combine components from kits with different lot numbers printed on the boxes and Certificates of Analysis If lots are mixed locus to locus imbalance and variation in signal intensity may occur 2 Determine the number of reactions to be set up This should include positive and negative control reactions Add 1 or 2 reactions to this number to compensate for pipetting error While this approach does consume a small amount of each reagent it ensures that you will have enough PCR amplification mix for all samples It also ensures that each reaction contains the same PCR amplification mix 3 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 8 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD022 Revised 9 15 www promega com 4 A Amplification of Extracted DNA continued 4 Add the final volume of each reagent listed in Table 1 to a sterile tube Note In tests performed at Promega we have found that reactions can remain at room temperature for up to 8 hours after reaction assembly and prior to thermal cycling with no adverse effect on amplificat
31. 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 final volume to 1 liter with deionized water Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 81 www promega com TMD022 Revised 9 15 9 F DNA Extraction and Quantitation Methods and Automation Support Promega offers a wide variety of reagents and automated methods for sample preparation DNA purification and DNA quantitation prior to STR amplification For analysis of database reference and other single source samples we recommend direct amplification from FTA punches or preprocessing of swabs and nonFTA punches with the SwabSolution Kit or PunchSolution Kit The SwabSolution Kit Cat DC8271 contains reagents for rapid DNA preparation from buccal swab samples prior to amplification The procedure lyses cells contained on the swab head and releases into solution sufficient DNA for STR amplification A small volume of the final 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 amplification For casework or samples that require DNA purification we recommend the DNA IQ System Cat DC6700 which is a DNA isolation system designed spec
32. capillaries viewer window in the data collection software Each injection will take approximately 45 minutes 30 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD022 Revised 9 15 www promega com 5 C Detection of Amplified Fragments Using the ABI PRISM 3100 Genetic Analyzer with Data Collection Software Version 1 0 1 or 1 1 Materials to Be Supplied by the User 95 C dry heating block water bath or thermal cycler crushed ice or ice water bath centrifuge compatible with 96 well plates aerosol resistant pipette tips 3100 capillary array 36cm performance optimized polymer 4 POP 4 for the 3100 10X genetic analyzer buffer with EDTA MicroAmp optical 96 well plate and septa for the 3100 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 w
33. internal size standard and is not a matter of concern 6 Double click on the Allelic Ladders macro A plots window will open to display the blue fluorescein dye allelic ladders i e Penta E D18S51 D21S11 TH01 and D3S1358 green JOE dye allelic ladders i e Penta E CSF1PO D16S539 D7S820 D13S317 and D5S818 and yellow TMR dye allelic ladders i e FGA TPOX D8S1179 vWA and Amelogenin Confirm that the correct allele designations were assigned to the allelic ladders Figure 24 in Section 6 J Note The software uses one ladder sample to determine allele sizes The macro uses the first ladder sample imported for allele designations If the POWER macro is run a second time the software will use the second ladder if the POWER macro is run a third time the software will use the third ladder etc until all ladders in the project are used If an allelic ladder fails to be analyzed or if many off ladder alleles are found in the samples samples should be re analyzed using another ladder from the project 7 Double click on the Display Fluorescein Data macro to display the blue dye for all sample injections Scroll down to observe and edit as needed 8 Double click on the Display TMR Data macro to display the yellow dye for all sample injections Scroll down to observe and edit as needed 9 Double click on the Display JOE Data macro to display the green dye for all sample injections Scroll down to observe and edit as needed
34. is linked to the plate the plate graphic will change from yellow to green the plate record moves from the pending plate records table to the linked plate records table and the Run Instrument button becomes enabled 32 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD022 Revised 9 15 www promega com 5 C Detection of Amplified Fragments Using the ABI PRISM 3100 Genetic Analyzer with Data Collection Software Version 1 0 1 or 1 1 continued 17 Select Run Instrument on the toolbar to start the sample run 18 Monitor electrophoresis by observing the run status array and capillary views windows in the collection software Each injection will take approximately 45 minutes 5 D Detection of Amplified Fragments Using the ABI PRISM 310 Genetic Analyzer Materials to Be Supplied by the User 95 C dry heating block water bath or thermal cycler crushed ice or ice water bath 310 capillaries 47cm 50 m performance optimized polymer 4 POP 4 10X genetic analyzer buffer with EDTA sample tubes and septa aerosol resistant pipette tips Hi Di formamide Applied Biosystems Cat 4311320 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 quali
35. many samples This may be due to changes in temperature or the CE column over time Use a different injection of allelic ladder to determine sizes in the PowerTyper 16 Macro Release 2 0 Do not use the first injection on a new column for the ladder sample The base pair size of alleles was incorrect because incorrect fragment sizes were assigned to the internal lane standard Confirm that internal lane standard fragment sizes are assigned correctly Re analyze the sample using GeneScan software and redefine the internal lane standard fragments Promega Corporation 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 TMD022 Revised 9 15 8 References 1 Edwards A et al 1991 DNA typing with trimeric and tetrameric tandem repeats Polymorphic loci detection systems and population genetics In The Second International Symposium on Human Identification 1991 Promega Corporation 31 52 2 Edwards A et al 1991 DNA typing and genetic mapping with trimeric and tetrameric tandem repeats Am J Hum Genet 49 746 56 3 Edwards A et al 1992 Genetic variation at five trimeric and tetrameric tandem repeat loci in four human population groups Genomics 12 241 53 4 Warne D et al 1991 Tetranucleotide repeat polymorphism at the human actin related pseudogene 2 actbp2 detected using the polymerase chain reaction
36. or shadow bands is due to the loss of a repeat unit during DNA amplification somatic variation within the DNA or both The amount of this artifact observed depends primarily on the locus and the DNA sequence being amplified Terminal nucleotide addition 17 18 occurs when Taq DNA polymerase adds a nucleotide generally adenine to the 3 ends of amplified DNA fragments in a template independent manner The efficiency with which this occurs varies with different primer sequences Thus an artifact band one base shorter than expected i e missing the terminal addition is sometimes seen We have modified primer sequences and added a final extension step of 60 C for 30 minutes 19 to the amplification protocols to provide conditions for essentially complete terminal nucleotide addition when recommended amounts of template DNA are used The presence of microvariant alleles alleles differing from one another by lengths other than the repeat length complicates interpretation and assignment of alleles There appears to be a correlation between a high degree of polymorphism a tendency for microvariants and increased mutation rate 20 21 Thus FGA and D21S11 display numerous relatively common microvariants For reasons yet unknown the highly polymorphic Penta E locus does not display frequent microvariants Table 4 70 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330
37. punch per 25 l amplification reaction Buccal samples on Bode Buccal DNA Collector devices Blood and buccal samples on nonFTA card punches e g S amp S 903 Pretreat these sample types with the PunchSolution Reagent Cat DC9271 to lyse nonFTA samples before adding the PCR amplification mix For more information see the PunchSolution Kit Technical Manual TMD038 Failure to pretreat these samples may result in incomplete profiles 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 7486MA 94 0 C 100 90 0 C 100 60 0 C 29 60 0 C 29 70 0 C 23 70 0 C 23 3 tmp 10 cycles 3 tmp 22 cycles Figure 2 The ramp rates for the GeneAmp PCR System 9700 thermal cycler Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 11 www promega com TMD022 Revised 9 15 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 a PCR amplification mix t
38. sensitive and must be stored in the dark We strongly recommend that pre amplification and post amplification reagents be stored and used separately with different pipettes tube racks etc For daily use the PowerPlex 16 HS 10X Primer Pair Mix and PowerPlex HS 5X Master Mix can be stored at 2 10 C for up to 1 week without loss of activity The Water Amplification Grade can be stored at 2 10 C long term Promega Corporation 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 TMD022 Revised 9 15 Available Separately P R O D U C T S I Z E C AT PowerTyper Macros Release 2 0 1 CD ROM DG3470 Not For Medical Diagnostic Use The PowerTyper Macros Release 2 0 for use with Genotyper software are available from Promega This CD ROM contains the file PowerTyper 16 Macro Release 2 0 for use with the PowerPlex 16 HS System The macros also can be downloaded at www promega com resources tools powertyper macros The proper panels and bins text files for use with GeneMapper ID and ID X software can be obtained from the Promega web site at www promega com resources tools genemapper id software panels and bin sets Matrix standards are required for initial setup of the color separation matrix The matrix standards are sold separately and are available for the ABI PRISM 310 Genetic Analyzer PowerPlex Ma
39. start is selected for the thermal cycling run A Select Method Options screen appears Select 9600 ramp mode and enter the reaction volume 3 After completion of the thermal cycling protocol store amplified samples at 20 C in a light protected box Note Long term storage of amplified samples at 4 C or higher may produce artifacts 14 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD022 Revised 9 15 www promega com 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 workflow 2 Depending on your preferred protocol place one or two 1 2mm storage card punches containing a buccal sample or one 1 2mm punch of a storage card containing whole blood in each well of a reaction plate 3 Prepare three identical reaction plates with punches from the same samples 4 Amplify samples using the thermal cycling protocol provided above but subject each plate to a different cycle number 5 Following amplification use your laboratory s validated separation and detection protocols to determine the optimal cycle number for the sample type and
40. vWA 16 16 17 18 17 17 16 19 Amelogenin X X X X X Y X Y Penta D 9 13 12 12 8 12 12 13 CSF1PO 9 10 10 12 10 11 12 12 12 D16S539 11 12 11 12 11 11 9 13 D7S820 9 11 10 11 11 11 8 11 D13S317 8 8 11 11 11 11 9 11 D5S818 11 12 11 11 11 13 12 12 1Information about strains 9947A and 9948 is available online at http ccr coriell org Sections Search Sample_Detail aspx Ref GM09947 and http ccr coriell org Sections Search Sample_Detail aspx Ref GM09948 Strain K562 is available from the American Type Culture Collection www atcc org Manassas VA Information about the use of 9947A and 9948 DNA as standard DNA templates can be found in reference 25 2Strain K562 displays three alleles at the D21S11 locus 3Strain 9948 displays three alleles at the CSF1PO locus The peak height for allele 12 is much lower than those for alleles 10 and 11 Promega Corporation 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 TMD022 Revised 9 15 9 B Power of Discrimination The fifteen STR loci amplified with the PowerPlex 16 HS System provide powerful discrimination Population statistics for these loci are displayed in Table 5 These data were generated as part of a collaboration 26 with The Bode Technology Group Springfield VA North Carolina Bureau of Investigation Raleigh NC Palm B
41. 0 Genetic Analyzer generate a new matrix and apply it to the samples For the ABI PRISM 3100 and 3100 Avant Genetic Analyzers and Applied Biosystems 3130 3130xl 3500 and 3500xL Genetic Analyzers perform a new spectral calibration and re run the samples Instrument sensitivities can vary Optimize the injection conditions See Section 5 Long term storage of amplified sample in formamide can result in degradation Repeat sample preparation using fresh formamide Promega Corporation 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 TMD022 Revised 9 15 Symptoms Possible Causes and Comments Extra peaks visible in one or all color channels continued 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 Allelic ladder not running the same as samples Allelic ladder and primer pair mix were not compatible Ensure 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
42. 4 203 21 15 Schlotterer C and Tautz D 1992 Slippage synthesis of simple sequence DNA Nucleic Acids Res 20 211 5 16 Smith J R et al 1995 Approach to genotyping errors caused by nontemplated nucleotide addition by Taq DNA polymerase Genome Res 5 312 7 17 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 18 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 19 Moller A Meyer E and Brinkmann B 1994 Different types of structural variation in STRs HumFES FPS HumVWA and HumD21S11 Int J Leg Med 106 319 23 68 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD022 Revised 9 15 www promega com 8 References continued 20 Brinkmann B Moller A and Wiegand P 1995 Structure of new mutations in 2 STR systems Int J Leg Med 107 201 3 21 Griffiths R et al 1998 New reference allelic ladders to improve allelic designation in a multiplex STR system Int J Legal Med 111 267 72 22 B r W et al 1997 DNA recommendations Further report of the DNA Commission of the ISFH regarding the use of short tandem r
43. 50 is required for spectral calibration on the ABI PRISM 3100 and 3100 Avant Genetic Analyzers and Applied Biosystems 3130 3130xl 3500 and 3500xL Genetic Analyzers The PowerPlex Matrix Standards 310 Cat DG4640 cannot be used to generate a matrix on these instruments For protocols and additional information on matrix standardization see the PowerPlex Matrix Standards 310 Technical Bulletin TBD021 For protocols and additional information on spectral calibration see the PowerPlex Matrix Standards 3100 3130 Technical Bulletin TBD022 These manuals are available online at www promega com resources protocols 4 Protocols for DNA Amplification Using the PowerPlex 16 HS System The PowerPlex 16 HS System is optimized for the GeneAmp PCR System 9700 thermal cycler An amplification protocol for the GeneAmp PCR Systems 9600 thermal cycler also is provided for extracted DNA The use of gloves and aerosol resistant pipette tips is highly recommended to prevent cross contamination Keep all pre amplification and post amplification reagents in separate rooms Prepare amplification reactions in a room dedicated for reaction setup Use equipment and supplies dedicated for amplification setup Meticulous care must be taken to ensure successful amplification A guide to amplification troubleshooting is provided in Section 7 A The concentration of 2800M Control DNA was determined by measuring absorbance at 260nm Quantificati
44. 726TA Figure 19 The Size Standard Editor Promega Corporation 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 TMD022 Revised 9 15 6 Choose Red for the Size Standard Dye 7 Enter the sizes of the internal lane standard fragments 60 80 100 120 140 160 180 200 225 250 275 300 325 350 375 400 425 450 475 500 550 and 600 bases See Section 9 D Figure 28 Note Definition and detection of the 600bp fragment is optional 8 Select OK 6 G Creating a Casework Analysis Method with GeneMapper ID Software Version 3 2 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 5 In the Analysis Method Editor enter a descriptive name for the analysis method such as PowerPlex16HS advanced 46 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD022 Revised 9 15 www promega com 6 G Creating a Casework Analysis Method with GeneMap
45. 8 30 Sprecher C J et al 1996 General approach to analysis of polymorphic short tandem repeat loci BioTechniques 20 266 76 31 Lins A M et al 1996 Multiplex sets for the amplification of polymorphic short tandem repeat loci silver stain and fluorescent detection BioTechniques 20 882 9 32 Jones D A 1972 Blood samples Probability of discrimination J Forensic Sci Soc 12 355 9 33 Brenner C and Morris J W 1990 In Proceedings from the International Symposium on Human Identification 1989 Promega Corporation 21 53 34 Mandrekar P V Krenke B E and Tereba A 2001 DNA IQ The intelligent way to purify DNA Profiles in DNA 4 3 16 35 Gill P Jeffreys A J and Werrett D J 1985 Forensic application of DNA fingerprints Nature 318 577 9 36 Krenke B E et al 2005 Development of a novel fluorescent two primer approach to quantitative PCR Profiles in DNA 8 1 3 5 37 Krenke E et al 2008 Developmental validation of a real time PCR assay for the simultaneous quantification of total human and male DNA Forensic Sci Int Genet 3 14 21 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 69 www promega com TMD022 Revised 9 15 9 Appendix 9 A Advantages of Using the Loci in the PowerPlex 16 HS System The loci included in the PowerPlex 16 HS Syste
46. Biosystems Cat 4410228 POP 4 polymer for the Applied Biosystems 3500 or 3500xL Genetic Analyzer anode buffer container cathode buffer 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 18 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD022 Revised 9 15 www promega com 5 A Detection of Amplified Fragments Using the Applied Biosystems 3500 or 3500xL Genetic Analyzer continued Sample Preparation 1 Prepare a loading cocktail by combining and mixing Internal Lane Standard 600 and Hi Di formamide as follows 0 5 l ILS 600 samples 9 5 l Hi Di formamide samples Note The volume of in
47. If peak heights are too high we recommend altering the loading cocktail to contain 0 25 l of ILS 600 and 9 75 l of formamide 3 Vortex for 10 15 seconds to mix 4 Pipet 10 l of formamide internal lane standard mix into each well 5 Add 1 l of amplified sample or 1 l of PowerPlex 16 HS Allelic Ladder Mix 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 data collection software to modify the injection time or voltage in the run module If peak heights are higher than desired use less DNA template in the amplification reactions or reduce the number of cycles in the amplification program by 2 4 cycles to achieve the desired signal intensity 6 Centrifuge plate briefly to remove air bubbles from the wells 7 Denature samples at 95 C for 3 minutes then immediately chill on crushed ice or in an ice water bath for 3 minutes Denature samples just prior to loading the instrument Promega Corporation 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 TMD022 Revised 9 15 Instrument Preparation Refer to the instrument user s manual for instructions on cleaning installing the capillary array performing a spatial calibration and adding polymer An
48. Nucleic Acids Res 19 6980 5 Ausubel F M et al 1996 Unit 15 The polymerase chain reaction In Current Protocols in Molecular Biology Vol 2 John Wiley and Sons NY 6 Sambrook J Fritsch E F and Maniatis T 1989 Chapter 14 In vitro amplification of DNA by the polymerase chain reaction In Molecular Cloning A Laboratory Manual Second Edition Cold Spring Harbor Laboratory Press Cold Spring Harbor New York 7 PCR Technology Principles and Applications for DNA Amplification 1989 Erlich H A ed Stockton Press New York NY 8 PCR Protocols A Guide to Methods and Applications 1990 Innis M A et al eds Academic Press San Diego CA 9 Butler J M 2005 Forensic DNA Typing 2nd ed Elsevier Academic Press London 10 Presley L A et al 1992 The implementation of the polymerase chain reaction PCR HLA DQ alpha typing by the FBI laboratory In The Third International Symposium on Human Identification 1992 Promega Corporation 245 69 11 Hartmann J M et al 1991 Guidelines for a quality assurance program for DNA analysis Crime Laboratory Digest 18 44 75 12 Internal Validation of STR Systems Reference Manual GE053 Promega Corporation 13 Kline M C et al 2005 Results from the NIST 2004 DNA quantitation study J Forensic Sci 50 570 8 14 Levinson G and Gutman G A 1987 Slipped strand mispairing A major mechanism for DNA sequence evolution Mol Biol Evol
49. Processing Data for Databasing and Paternity Samples 1 Select File then New Project 2 Select Edit then Add Samples to Project 3 Browse to the location of the run files Highlight desired files 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 5 In the Analysis Method column select the analysis method created above 6 In the Panel column select the panels text file that was imported in Section 6 A 42 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD022 Revised 9 15 www promega com 6 D Creating a Databasing or Paternity Analysis Method with GeneMapper ID X Software Version 1 2 continued 7 In the Size Standard column select the size standard that was created in Section 6 B 8 If analyzing data from an ABI PRISM 310 Genetic Analyzer ensure that the appropriate matrix file is selected in the Matrix column 9 Select Analyze green arrow button to start data analysis Note By default the software displays the Analysis Requireme
50. Revised 9 15 TMD022 T E C H N I C A L M A N U A L PowerPlex 16 HS System Instructions for Use of Products DC2100 and DC2101 Promega Corporation 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 TMD022 Revised 9 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 16 HS System 1 Description 2 2 Product Components and Storage Conditions 4 3 Before You Begin 5 3 A Precautions 5 3 B Matrix Standardization or Spectral Calibration 6 4 Protocols for DNA Amplification Using the PowerPlex 16 HS System
51. X software 2 Select Tools then select 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 file imported in the Getting Started Section Select the file then Import 6 In the navigation pane highlight the PowerPlex 16 panels folder that you just imported in Step 5 7 Select File then Import Bin Set 8 Navigate to the bins text file that was imported in the Getting Started Section Select the file then Import 9 In the navigation pane highlight the PowerPlex 16 panels folder that you just imported in Step 5 10 Select File then Import Marker Stutter to import the marker specific stutter ratio for the panels and bins text files A warning box will appear asking if you want to overwrite current values Select Yes 11 Navigate to the stutter file that was imported in the Getting Started Section Select the file then Import 12 At the bottom of the Panel Manager window select OK This will save the panels bins and stutter text files and close the window Promega Corporation 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 TMD022 Revised 9 15 6 B Creating a Size Standard with GeneMapper ID X Software Version 1 2 1 Select Tools
52. al extraction This system uses a standard proteinase K digestion and a combination of phase separation and differential centrifugation to separate sperm and epithelial DNA quickly and easily The Differex System offers similar recovery as the standard method commonly used for differential extraction The Differex System in combination with the DNA IQ System can be automated to extract up to 48 differential extractions in less than 5 hours including incubation time and less than 1 hour of hands on laboratory time 82 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD022 Revised 9 15 www promega com 9 F DNA Extraction and Quantitation Methods and Automation Support continued For applications requiring human specific DNA quantification the Plexor HY System Cat DC1000 was developed 37 38 See Section 9 G for ordering information For information about automation of Promega chemistries on automated workstations using Identity Automation solutions contact your local Promega Branch Office or Distributor contact information available at www promega com support worldwide contacts e mail genetic promega com or visit www promega com idautomation 9 G Related Products STR Systems Product Size Cat PowerPlex 16 Monoplex System Penta E Fluorescein 100 reactions DC6591 PowerPlex 16 Monoplex System Penta D
53. alyze 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 New Select Regular in the Type drop down list and select HIDFragmentAnalysis36_POP4 in the Template drop down list Confirm that the injection time is 5 seconds and the injection voltage is 3kV Lengthen the run time to 1 800 seconds Give a descriptive name to your run module and select OK Note Instrument sensitivities can vary The injection time and voltage may be adjusted in the Module Manager A suggested range for the injection time is 3 22 seconds and for the injection voltage is 1 3kV 2 In the Protocol Manager select New Type a name for your protocol Select Regular in the Type drop down list and select the run module you created in the previous step in the Run Module drop down list Lastly select F in the Dye Set drop down list Select OK 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 autoanal
54. arately from those used following amplification PowerPlex 16 HS Allelic Ladder Mix and Internal Lane Standard 600 Always include a negative control reaction i e no template to detect reagent contamination We highly recommend the use of gloves and aerosol resistant pipette tips 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 6 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD022 Revised 9 15 www promega com 3 B Matrix Standardization or Spectral Calibration Proper spectral calibration is critical to evaluate multicolor systems with the ABI PRISM 310 3100 and 3100 Avant Genetic Analyzers and Applied Biosystems 3130 3130xl 3500 and 3500xL Genetic Analyzers A matrix must be generated for each individual instrument The PowerPlex Matrix Standards 310 Cat DG4640 is required for matrix standardization for the ABI PRISM 310 Genetic Analyzer The PowerPlex Matrix Standards 3100 3130 Cat DG4650 cannot be used to generate a matrix on the ABI PRISM 310 Genetic Analyzer The PowerPlex Matrix Standards 3100 3130 Cat DG46
55. ate 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 Off ladder alleles An allelic ladder from a different 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 C or 6 D Panels text file selected for analysis was incorrect for the STR system used Assign correct panels text file that corresponds to the STR system used for amplification The allelic ladder was not identified as an allelic ladder in the Sample Type column The internal lane standard was not properly identified in the sample Manually redefine 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 An allelic ladder from a different run than the samples was used Re analyze samples with an allelic ladder from the same run Size standard not called correctly Starting data point was incorrect for the partial range chosen in Section 6 D Adjust the starting data point in th
56. cation type dye set capillary length polymer run module and appropriate protocol information The only settings that were changed from the default settings are dye set and run time When creating an Instrument Protocol be sure to select the same dye set that was used to perform the Promega 4 dye spectral calibration We recommend using a run time of 1 500 seconds and the default injection conditions Run time and other instrument settings should be optimized and validated in your laboratory Assign a descriptive protocol name Note For more detailed information refer to the Applied Biosystems 3500 3500xL Genetic Analyzers User Guide 9248TA Figure 4 The Create New Instrument Protocol window Promega Corporation 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 TMD022 Revised 9 15 3 To create a new Size Standard for the QC protocol navigate to the Library Select Size Standards then select Create Alternatively a previously created Size Standard may be used Assign the size standard the name ILS600 or another appropriate name Choose Red as the Dye Color The fragments in the size standard are 60 80 100 120 140 160 180 200 225 250 275 300 325 350 375 400 425 450 475 500 550 and 600 bases See Figure 5 Note Definition and detection of the 600bp fragment is optional 9249TA Figur
57. ction 9 A Promega Corporation 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 TMD022 Revised 9 15 6 J Results Representative results of the PowerPlex 16 HS System are shown in Figure 23 The PowerPlex 16 HS Allelic Ladder Mix is shown in Figure 24 A B C D 7918TA Figure 23 The PowerPlex 16 HS System A single source template DNA 0 5ng was amplified using the PowerPlex 16 HS System Amplification products were mixed with Internal Lane Standard 600 and analyzed with an Applied Biosystems 3130 Genetic Analyzer using a 3kV 5 second injection Results were analyzed using GeneMapper ID software version 3 2 Panel A An electropherogram showing the peaks of the fluorescein labeled loci D3S1358 TH01 D21S11 D18S51 and Penta E Panel B An electropherogram showing the peaks of the JOE labeled loci D5S818 D13S317 D7S820 D16S539 CSF1PO and Penta D Panel C An electropherogram showing the peaks of the TMR labeled loci Amelogenin vWA D8S1179 TPOX and FGA Panel D An electropherogram showing the 60bp to 500bp fragments of the Internal Lane Standard 600 52 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD022 Revised 9 15 www promega com 6 J Results continued Figure 24 The PowerPlex 16 HS Allelic Ladd
58. e Poor capillary electrophoresis injection ILS 600 peaks also affected Re inject the sample Check the syringe or pump system for leakage Check the laser power Samples were not denatured completely Heat denature samples for the recommended time then cool on crushed ice or in an ice water bath immediately prior to 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 quality formamide was used Use only Hi Di formamide when analyzing samples 54 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD022 Revised 9 15 www promega com 7 A Amplification and Fragment Detection continued Symptoms Possible Causes and Comments Extra peaks visible in one or all color channels Contamination with another template DNA or previously amplified DNA Cross contamination can be a problem Use aerosol resistant pipette tips and change gloves regularly Samples were not denatured completely Heat denature samples for the recommended time and cool on crushed ice or in an ice water bath immediately prior to 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 Artifacts of STR amplification Amplification of STRs can result in artifacts that appear as faint peaks one repeat unit s
59. e 5 The Create New Size Standard window 22 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD022 Revised 9 15 www promega com 5 A Detection of Amplified Fragments Using the Applied Biosystems 3500 or 3500xL Genetic Analyzer continued 4 To create a new QC Protocol navigate to the Library Select QC Protocols then select Create A previously created QC Protocol may be used Assign a descriptive protocol name Select the size standard created in Step 3 The settings for the QC protocol should be based on the internally validated conditions for the PowerPlex 16 HS System on the Applied Biosystems 3500 or 3500xL Genetic Analyzer Figure 6 shows one option for these settings 9250TA Figure 6 The Create New QC Protocol window Promega Corporation 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 TMD022 Revised 9 15 5 To create a new Assay navigate to the Library Select Assays then select Create Alternatively a previously created Assay may be used In the Create New Assay window Figure 7 select the instrument protocol created in Step 2 and the QC protocol created in Step 4 Assign a descriptive assay name Select the application type HID An Assay is required for all named samples on a plate
60. e 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 ILS 600 peaks defined in the size standard were detected during the run Create a new size standard using the internal lane standard fragments present in the sample Re run samples using a longer run time Peaks in size standard missing If peaks are below threshold decrease the peak amplitude threshold in the analysis method for the red channel to include peaks If peaks are low quality redefine the size standard for the sample to skip these peaks 62 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD022 Revised 9 15 www promega com 7 D GeneMapper ID X Software continued Symptoms Possible Causes and Comments Significantly raised baseline Poor spectral calibration for the ABI PRISM 3100 and 3100 Avant Genetic Analyzers and Applied Biosystems 3130 3130xl 3500 and 3500xL Genetic Analyzers Perform a new spectral calibration and re run the samples Poor matrix for the ABI PRISM 310 Genetic Analyzer Re run and optimize the matrix Make sure that the matrix applied was generated on the same instrument
61. e in USA 800 356 9526 608 274 4330 Fax 608 277 2516 27 www promega com TMD022 Revised 9 15 18 In the Run Information window Figure 12 assign a Run Name Select Start Run not shown 9256TA Figure 12 Assigning a run name 5 B Detection of Amplified Fragments Using 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 or ice water bath centrifuge compatible with 96 well plates aerosol resistant pipette tips 3100 or 3130 capillary array 36cm performance optimized polymer 4 POP 4 for the 3100 or 3130 10X genetic analyzer buffer with EDTA MicroAmp optical 96 well plate and septa Hi Di formamide Applied Biosystems Cat 4311320 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
62. e size standard were detected during the run Definition and detection of the 600bp fragment is optional Create a new size standard using the internal lane standard fragments present in the sample Re run samples using a longer run time 5686TA Figure 26 An example showing improper assignment of size standard fragments in the GeneMapper ID Software 64 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD022 Revised 9 15 www promega com 7 E GeneMapper ID Software continued Symptoms Possible Causes and Comments Peaks in size standard missing If peaks are below threshold decrease the peak amplitude threshold in the analysis method for the red channel to include peaks If peaks are low quality redefine the size standard for the sample to skip these peaks Error message Either panel size standard or analysis method is invalid The size standard and analysis method were not in the same mode Classic vs Basic or Advanced Be sure both files are set to the same mode either Classic or Basic or Advanced mode No alleles called but no error Panels text file was not selected for sample In the Panel column select the appropriate panels text file for the STR system that was used No size standard was selected In the Size Standard column be sure to select the appropriate size standard Size
63. e the plate record spreadsheet for the wells you have loaded Enter appropriate information into the Sample Name and Color Info columns For allelic ladder samples insert the word ladder into the Color Info column for the blue yellow and green dye colors This information must be entered to successfully analyze data with the PowerTyper 16 Macro Release 2 0 8 In the BioLIMS Project column select 3100_Project1 from the drop down menu 9 In the Dye Set column select Z from the drop down menu 10 When using the ABI PRISM 3100 Data Collection Software Version 1 0 1 or 1 1 select GeneScan36_POP4PowerPlex16_3kV_11secs_1800 from the drop down menu in the Run Module 1 column 11 To collect data without autoanalyzing select No Selection in the Analysis Module 1 column Analysis parameters can be applied after data collection and during data analysis using the GeneScan software 12 Select OK This new plate record will appear in the pending plate records table on the plate setup page of the collection software 13 Place samples in the instrument and close the instrument doors 14 Locate the pending plate record that you just created and click once on the name 15 Once the pending plate record is highlighted click on the plate graphic that corresponds to the plate on the autosampler that contains your amplified samples to link the plate to the plate record 16 When the plate record
64. e thresholds are the minimum peak heights at which the software will call a peak Values for peak amplitude thresholds are usually 50 200RFU and should be determined by individual laboratories 4 The analysis parameters can be saved in the Params folder 5 Apply the stored analysis parameters file to the samples 6 Assign a new size standard Select a sample file highlight the arrow next to size standard then select define new Assign the size standard peaks as shown in Figure 28 in Section 9 D Store the size standard in the Size Standards folder 7 Apply the size standard file to the samples then analyze the sample files See below for additional information on the use of the PowerTyper 16 Macro Release 2 0 and Genotyper software For additional information regarding the GeneScan software refer to the GeneScan Analysis Software User s Manual Notes 1 Peak heights outside the linear range of the instrument may generate artifact peaks due to instrument saturation i e overloading the sample Bleedthrough pull ups from one color to another may be observed Saturated signal also may appear as two peaks split peak 2 If peak heights are not within the linear range of detection of the instrument the ratio of stutter peaks to real allele peaks increases and allele designations become difficult to interpret The balance of peak heights also may appear less uniform Promega Corporation 2800 Woods H
65. e usually 50 150RFU and should be determined by individual laboratories 11 Select the Peak Quality tab You may change the settings for peak quality Note For Steps 11 and 12 see the GeneMapper ID user s manual for more information 5723TA Figure 21 The GeneMapper ID Peak Detector tab 12 Select the Quality Flags tab You may change these settings 13 Select OK to save your settings 48 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD022 Revised 9 15 www promega com 6 G 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 files Highlight the desired files 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 5 In the Analysis Method column select the analysis method created previously in this section 6 In the Panel column select the panels text file
66. each County Sheriff s Office West Palm Beach FL Virginia Division of Forensic Science Richmond VA and Charlotte Mecklenburg Police Department Laboratory NC Data generation included analysis of over 200 individuals from African American Caucasian American and Hispanic American populations Data for Asian Americans include analysis of more than 150 individuals For additional population data for STR loci see references 27 32 and the Short Tandem Repeat DNA Internet DataBase at www cstl nist gov div831 strbase Table 5 shows the matching probability 33 for the PowerPlex 16 HS System in various populations The matching probability of the PowerPlex 16 HS System ranges from 1 in 1 83 1017 for Caucasian Americans to 1 in 1 41 1018 for African Americans A measure of discrimination often used in paternity analyses is the paternity index PI a means for presenting the genetic odds in favor of paternity given the genotypes for the mother child and alleged father 34 The typical paternity indices for the PowerPlex 16 HS System are shown in Table 5 The PowerPlex 16 HS System provides typical paternity indices exceeding 500 000 in each population group An alternative calculation used in paternity analyses is the power of exclusion 34 This value calculated for the PowerPlex 16 HS System exceeds 0 999998 in all populations tested Table 5 Table 5 Matching Probabilities Paternity Indices and Power of Exclusion of the P
67. ear gloves and safety glasses when working with formamide Sample Preparation 1 Prepare a loading cocktail by combining and mixing Internal Lane Standard 600 and Hi Di formamide as follows 0 5 l ILS 600 samples 9 5 l Hi Di formamide samples Note The volume of internal lane standard used in the loading cocktail can be increased or decreased to adjust the intensity of the size standard peaks The optimal peak height for the 100 base fragment of the internal lane standard is 500 1 000RFU If peak heights are too low we recommend altering the formamide internal lane standard mix to contain 1 0 l of ILS 600 and 9 0 l of Hi Di formamide If peak heights are too high we recommend altering the loading cocktail to contain 0 25 l of ILS 600 and 9 75 l of formamide 2 Vortex for 10 15 seconds to mix 3 Pipet 10 l of formamide internal lane standard mix into each well 4 Add 1 l of amplified sample or 1 l of PowerPlex 16 HS Allelic Ladder Mix 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 increased or decreased Use the Module Manager in the data collection software to modify the injection time or voltage in the run module If peak heights are higher than desired use less DNA template in the amplification reactions or reduce the number of cycles in the
68. ease 2 0 3 Create a new GeneScan injection list Select the appropriate sample sheet from the drop down menu 4 Select the GS STR POP4 1ml F Module using the drop down menu Change the injection time to 3 seconds and the run time to 30 minutes Keep the settings for the remaining parameters as shown below Inj Secs 3 Inj kV 15 0 Run kV 15 0 Run C 60 Run Time 30 You may need to optimize the injection time for individual instruments Injection times of 2 5 seconds are suggested for samples that contain 0 5 1ng of template DNA Note Migration of fragments may vary slightly over the course of a long ABI PRISM 310 Genetic Analyzer run This may be due to changes in temperature or changes in the column When analyzing many samples injections of allelic ladder at different times throughout the run can aid in accurately genotyping samples 5 Select the appropriate matrix file see Section 3 B 6 To analyze data automatically select the auto analyze checkbox and the appropriate analysis parameters and size standard Refer to the ABI PRISM 310 Genetic Analyzer User Guide for specific information on these options 7 Enter an operator name 8 After loading the sample tray and closing the doors select Run to start the capillary electrophoresis system 9 Monitor electrophoresis by observing the raw data and status windows Each sample will take approximately 40 minutes for syringe pumping sample i
69. ed Biosystems user bulletin titled Installation Procedures and New Features for GeneMapper ID Software 3 2 5785TA Figure 22 The GeneMapper ID Allele tab with settings for using a 20 peak filter 10 Select the Peak Detector tab We recommend the settings shown in Figure 21 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 first defined 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 and should be determined by individual laboratories 11 Select the Peak Quality tab You may change the settings for peak quality Note For Steps 11 and 12 see the GeneMapper ID user s manual for more information 50 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD022 Revised 9 15 www promega com 6 H Creating a Databasing or Paternity Analysis Method with GeneMapper ID Software Version 3 2 continued 12 Select the Quality Flags tab You may change these settings 13 Select OK to save your settings Processing Data for Databasing or Paternity Samples
70. ele designations 78 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD022 Revised 9 15 www promega com Sample Analysis Using the Genotyper Software and PowerTyper 16 Macro continued 5 For casework double click on the POWER macro The POWER macro identifies alleles in the ladder sample and calculates offsets for all loci This process may take several minutes When completed a plots window will open to display the allelic ladders i e Penta E D18S51 D21S11 TH01 and D3S1358 Alternatively for databasing or paternity double click on the POWER 20 Filter macro This macro has a higher level of filtering than the standard POWER macro to reduce the need for manual editing of peak labels The POWER 20 Filter should not be used if mixtures may exist In general allelic ladders contain fragments of the same lengths as many known alleles for the locus Allelic ladder sizes and repeat units are listed in Table 3 Section 9 A Analysis using GeneScan and Genotyper software allows allele determination by comparing amplified sample fragments with allelic ladders and internal lane standards When using an internal lane standard the calculated lengths of allelic ladder components might differ from those listed in the table This is due to differences in migration resulting from sequence differences between the allelic ladder fragments and
71. elic ladder Promega recommends using GeneMapper ID software to analyze PowerPlex reactions If using GeneMapper ID software version 3 2 be sure that the analysis method selected is an HID method This can be verified by opening the analysis method using the GeneMapper Manager then selecting the General tab The analysis type cannot be changed If the method is not HID it should be deleted and a new analysis method created 7 F PowerTyper 16 Macro Symptoms Possible Causes and Comments File does not open on your computer Genotyper software was not installed Be certain that the Genotyper software version 2 5 Macintosh or version 3 6 or higher Windows NT is installed Incorrect version of Genotyper software The PowerTyper 16 Macro will not work with Genotyper software versions prior to version 2 5 Error message Could not complete the Run Macro command because no dye lanes are selected Allelic ladder sample files were not identified Be certain the Sample Info or Color Info column for each lane containing PowerPlex 16 HS Allelic Ladder Mix contains the word ladder The macro uses the word ladder to identify sample files containing allelic ladder All four dye colors were not imported For Genotyper software versions 2 5 and 3 5 or higher set preferences in the Edit menu to import the blue green yellow and red colors Error message Could not complete the Run
72. epeat systems Int J Legal Med 110 175 6 23 Gill P et al 1997 Considerations from the European DNA Profiling Group EDNAP concerning STR nomenclature Forensic Sci Int 87 185 92 24 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 25 Levadokou E N et al 2001 Allele frequencies for fourteen STR loci of the PowerPlex 1 1 and 2 1 multiplex systems and Penta D locus in Caucasians African Americans Hispanics and other populations of the United States of America and Brazil J Forensic Sci 46 736 61 26 Lins A M et al 1998 Development and population study of an eight locus short tandem repeat STR multiplex system J Forensic Sci 43 1168 80 27 Puers C et al 1993 Identification of repeat sequence heterogeneity at the polymorphic STR locus HUMTH01 AATG n and reassignment of alleles in population analysis using a locus specific allelic ladder Am J Hum Genet 53 953 8 28 Hammond H et al 1994 Evaluation of 13 short tandem repeat loci for use in personal identification applications Am J Hum Genet 55 175 89 29 Bever R A and Creacy S 1995 Validation and utilization of commercially available STR multiplexes for parentage analysis In Proceedings from the Fifth International Symposium on Human Identification 1994 Promega Corporation 61
73. er Mix The PowerPlex 16 HS Allelic Ladder Mix was analyzed with an Applied Biosystems 3130 Genetic Analyzer using a 3kV 5 second injection The sample file was analyzed with the GeneMapper ID software version 3 2 and PowerPlex 16 HS panels and bins text files Panel A The fluorescein labeled allelic ladder components and their allele designations Panel B The JOE labeled allelic ladder components and their allele designations Panel C The TMR labeled allelic ladder components and their allele designations Artifacts and Stutter Stutter products are a common amplification artifact associated with STR analysis 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 The pattern and intensity of stutter may differ slightly between primer sets for the same loci In addition to stutter peaks other artifact peaks can be observed at some of the PowerPlex 16 HS System loci Low level products can be seen in the n 2 and n 2 positions two bases below and above the true allele peak respectively with some loci such as D21S11 Samples may show low level artifacts in the regions between D21S11 and D18S51 D7S820 and D16S539 and D8S1179 and TPOX Occasionally an off ladder artifact can be seen in the 690 691bp position in the flu
74. evised 9 15 Symptoms Possible Causes and Comments Off ladder alleles An allelic ladder from a different 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 G or 6 H Panels text file selected for analysis was incorrect for the STR system used Assign correct panels text file that corresponds to the STR system used for amplification The allelic ladder was not identified as an allelic ladder in the Sample Type column The wrong analysis type was chosen for the analysis method Be sure to use the HID analysis type The internal lane standard was not properly identified in the sample Manually redefine the sizes of the size standard fragments in the sample Size standard not called correctly Figure 26 Starting data point was incorrect for the partial range chosen in Section 6 G Adjust the starting data point in the analysis method Alternatively use a full range for the analysis Extra peaks in advanced mode size standard Open the Size Match Editor Highlight the extra peak select Edit and select delete size label Select auto adjust sizes Run was too short and larger peaks in ILS were not captured Not all ILS 600 peaks defined in th
75. ew Results Group window 9254TA Figure 10 Defining plate properties 26 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD022 Revised 9 15 www promega com 5 A Detection of Amplified Fragments Using the Applied Biosystems 3500 or 3500xL Genetic Analyzer continued 10 Select Assign Plate Contents Figure 11 11 Assign sample names to wells 12 In the lower left portion of the screen under Assays use the Add from Library option to select the Assay created in Step 5 or one previously created Click on the Add to Plate button and close the window 13 Under File Name Convention use the Add from Library option to select the File Name Convention created in Step 6 or one previously created Click on the Add to Plate button and close the window 14 Under Results Groups use the Add from Library option to select the Results Group created in Step 7 or one previously created Click on the Add to Plate button and close the window 15 Highlight the sample wells then select the boxes in the Assays File Name Conventions and Results Groups that pertain to those samples 16 Select Link Plate for Run 17 The Load Plate window will appear Select Yes 9255TA Figure 11 Assigning plate contents Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Fre
76. extraction procedure used 3Apparent DNA concentrations can differ depending on the DNA quantification method used 13 The amount of DNA template recommended here is based on DNA concentrations determined by measuring absorbance at 260nm We strongly recommend that you perform experiments to determine the optimal DNA amount based on your DNA quantification method 5 Vortex the PCR amplification mix for 5 10 seconds then pipet PCR amplification mix into each reaction well Failure to vortex the PCR amplification mix sufficiently can result in poor amplification peak height imbalance and extra peaks 6 Add the template DNA 0 5ng for each sample to the respective well containing PCR amplification mix 7 For the positive amplification control vortex the tube of 2800M Control DNA then dilute an aliquot to 0 5ng in the desired template DNA volume Add 0 5ng of the diluted DNA to a reaction well containing PCR amplification mix 8 For the negative amplification control pipet Water Amplification Grade or TE 4 buffer instead of template DNA into a reaction well containing PCR amplification mix 9 Seal the plate Optional Briefly centrifuge the plate to bring contents to the bottom and remove any air bubbles Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 9 www promega com TMD022 Revised 9 15 Thermal Cycling Thi
77. he tube first then add PowerPlex HS 5X Master Mix PowerPlex 16 HS 10X Primer Pair Mix and 5X AmpSolution Reagent For FTA card punches the template DNA will be added at Step 6 5 Vortex the PCR amplification mix for 5 10 seconds then pipet 25 l of PCR amplification mix into each reaction well Failure to vortex the PCR amplification mix sufficiently can result in poor amplification or locus to locus imbalance 6 For FTA storage cards add one or two 1 2mm punches from a card containing a buccal sample 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 amplification mix to the pretreated punches Note It also is acceptable to add the FTA card punch first then add the PCR amplification mix 12 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD022 Revised 9 15 www promega com 4 B Direct Amplification of DNA from Storage Card Punches continued 7 For the positive amplification control add 1 l 10ng of the 2800M Control DNA to a reaction well containing 25 l of PCR amplification mix Notes 1 Do not include blank storage card punches in the positive control reactions 2 Optimization of the amount of 2800M Control DNA may be required based on thermal cycling conditions and laboratory preferences Typically 10ng of
78. her than desired use less DNA template in the amplification reactions or reduce the number of cycles in the amplification program by 2 4 cycles to achieve the desired signal intensity Promega Corporation 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 TMD022 Revised 9 15 4 Centrifuge tubes briefly to remove air bubbles from the wells 5 Denature samples by heating at 95 C for 3 minutes and immediately chill on crushed ice or in an ice water bath for 3 minutes Denature samples just prior to loading 6 Place tubes in the appropriate autosampler tray 7 Place the autosampler tray in the instrument and close the instrument doors Instrument Preparation Refer to the instrument user s manual for instructions on cleaning the pump block installing the capillary calibrating the autosampler and adding polymer to the syringe 1 Open the ABI PRISM 310 Data Collection Software 2 Prepare a GeneScan sample sheet as described in the ABI PRISM 310 Genetic Analyzer User Guide Enter the appropriate sample information in the Sample Info column For rows containing PowerPlex 16 HS Allelic Ladder Mix insert the word ladder in the Sample Info column for the blue dye color yellow dye color and green dye color This information must be entered to successfully analyze your data using the PowerTyper 16 Macro Rel
79. hes 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 amplification failure Amplification was inhibited when using more than one storage card punch with blood Use only one 1 2mm storage card punch with blood Make sure that the PCR amplification mix also contained AmpSolution Reagent Omission of AmpSolution Reagent from amplification reactions will result in amplification failure NonFTA card punches were not pretreated with PunchSolution Reagent Active PunchSolution Reagent carried over into the amplification reaction when using nonFTA card punches Ensure that the heat block was set at 70 C and samples were incubated for 30 minutes Incubation for shorter time periods may result in incomplete inactivation of the PunchSolution Reagent We have not tested longer incubation times Inactive PunchSolution Reagent Thaw the PunchSolution Reagent at 2 10 C Do not store reagents in the refrigerator door where the temperature can fluctuate Do not refreeze avoid multiple freeze thaw cycles as this may reduce activity Faint or absent peaks for the positive control reaction If the positive control reaction failed to amplify check to make sure that the correct amount of 2800M Control DNA was added to the reaction Do not include a blank punch in the positive control reaction Presence of a blank p
80. ifically for forensic samples 35 This system uses paramagnetic particles to prepare clean samples for STR analysis easily and efficiently and can be used to extract DNA from stains or liquid samples such as blood or solutions The DNA IQ Resin eliminates PCR inhibitors and contaminants frequently encountered in casework samples With larger samples the DNA IQ System delivers a consistent amount of total DNA The system has been used to isolate and quantify DNA from routine sample types including buccal swabs stains on FTA paper and liquid blood Additionally DNA has been isolated from casework samples such as tissue differentially separated sexual assault samples and stains on support materials The DNA IQ System has been tested with the PowerPlex Systems to ensure a streamlined process See Section 9 G for ordering information To process sexual assault samples differential extraction can be used to enrich sperm cells in the presence of an excess of epithelial cells 36 Traditionally these samples are processed by performing a controlled lysis of epithelial cells in the absence of a reducing agent and centrifuging samples to separate the pellet of intact sperm and cell debris from the buffer containing DNA from lysed epithelial cells However this method is time consuming and labor intensive and several washings and centrifugations often are required to obtain sperm free of epithelial DNA The Differex System simplifies differenti
81. ification was inhibited when using more than one storage card punch with blood Use only one 1 2mm storage card punch with blood 58 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD022 Revised 9 15 www promega com 7 B Direct Amplification from Storage Card Punches continued Symptoms Possible Causes and Comments Peak height imbalance continued Active PunchSolution Reagent carried over into the amplification reaction Larger loci are most susceptible to carryover and will drop out before the smaller loci Ensure that the heat block was set at 70 C and samples were incubated for 30 minutes Incubation for shorter time periods may result in incomplete inactivation of the PunchSolution Reagent Using a smaller amplification reaction volume may compromise performance when using 10 l of PunchSolution Reagent Reducing the PunchSolution Reagent volume may improve results for reactions with reduced amplification volumes Optimization and validation are required Inactive PunchSolution Reagent Thaw the PunchSolution Reagent at 2 10 C Do not store reagents in the refrigerator door where the temperature can fluctuate Do not refreeze avoid multiple freeze thaw cycles as this may reduce activity Carryover of excess PunchSolution Reagent into amplification reaction We recommend treating one 1 2mm nonFTA ca
82. ine internal lane standard fragments and re analyze the sample using GeneScan software Compare the size of the smallest allele in the allelic ladder with the base pair size and range listed in the categories for the same alleles If necessary increase the category start range in the category window to greater than 6bp and save the macro under a new name Allelic ladder peaks were too high causing stutter peaks to be called as allele peaks Use a shorter injection time decrease the amount of allelic ladder used or re analyze the allelic ladder sample using increased peak amplitude thresholds in the GeneScan analysis parameters Allelic ladder data were not compatible with the PowerTyper Macro file used Confirm that the PowerTyper Macro file matches the allelic ladder being used The plots window or allele table does not display all data The macros were not run in the proper order Use the POWER or POWER 20 Filter macro option All four dye colors were not imported For Genotyper software versions 2 5 and 3 5 or higher set preferences in the Edit menu to import the blue green yellow and red colors The Check ILS macro displays an empty plot window All four dye colors were not imported For Genotyper software versions 2 5 and 3 5 or higher set preferences in the Edit menu to import the blue green yellow and red colors Off ladder peaks Migration of samples changed slightly over the course of a CE run with
83. ing into the reaction tubes or plate Tubes of 5X Master Mix and 10X Primer Pair Mix from different lots were used The PowerPlex HS 5X Master Mix and PowerPlex 16 HS 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 56 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD022 Revised 9 15 www promega com 7 B Direct Amplification from Storage Card Punches The following information is specific to direct amplification of DNA from storage card punches For information about general amplification and detection see Section 7 A Symptoms Possible Causes and Comments Faint or absent allele peaks The reaction volume was too low This system is optimized for a final reaction volume of 25 l to overcome inhibitors present in storage cards Decreasing the reaction volume may result in suboptimal performance especially when amplifying DNA on storage card punches directly Poor sample deposition Shedding and collection of donor cells was variable Increase cycle number Poor sample transfer to storage card or variable sampling from the storage card Take punches from a different portion of the card Increasing cycle number also can improve low peak heights Too much sample in the reaction Use one or two 1 2mm storage card punc
84. ion results Amplification of gt 1 0ng of DNA template may result in an imbalance in peak heights from locus to locus The smaller loci show greater amplification yield than the larger loci Reducing the number of cycles in the amplification program by 2 to 4 cycles i e 10 20 or 10 18 cycling can improve locus to locus balance Table 1 PCR Amplification Mix for the PowerPlex 16 HS System PCR Amplification Mix Component1 Volume Per Reaction Number of Reactions Final Volume Water Amplification Grade to a final volume of 25 0 l PowerPlex HS 5X Master Mix 5 0 l PowerPlex 16 HS 10X Primer Pair Mix 2 5 l template DNA 0 5 1ng 2 3 up to 17 5 l total reaction volume 25 l 1Add Water Amplification Grade to the tube first then add PowerPlex HS 5X Master Mix and PowerPlex 16 HS 10X Primer Pair Mix The template DNA will be added at Step 6 2Store DNA templates in nuclease free water or TE 4 buffer 10mM Tris HCl pH 8 0 0 1mM EDTA If the DNA template is stored in TE buffer that is not pH 8 0 or contains a higher EDTA concentration the volume of DNA added should not exceed 20 of the final reaction volume Amplification efficiency and quality can be altered greatly by changes in pH due to added Tris HCl available magnesium concentration due to chelation by EDTA or PCR inhibitors which may be present at low concentrations depending on the source of the template DNA and the
85. ll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD022 Revised 9 15 www promega com 6 C Creating a Casework Analysis Method with GeneMapper ID X Software Version 1 2 continued 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 on the ABI PRISM 310 3100 and 3100 Avant Genetic Analyzers and Applied Biosystems 3130 and 3130xl Genetic Analyzers but should be determined by individual laboratories 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 peak amplitude thresholds from internal validation studies 3 The normalization box can be checked regardless of whether normalization was or was not applied during data collection 11 Select the Peak Quality tab You may change the settings for peak quality Note For Steps 11 and 12 see the GeneMapper ID X user s manual for more information 12 Select the SQ amp GQ Settings tab You may change these settings 13 Select Save to save the new analysis method 14 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
86. llele 2 Category Allele 1 Category Allele 2 Category Allele 1 Category Allele 2 Category Allele 1 Category Allele 2 CODIS Table Format Sample Info Category Peak 1 Peak 2 80 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD022 Revised 9 15 www promega com 9 D The Internal Lane Standard 600 The Internal Lane Standard ILS 600 contains 22 DNA fragments of 60 80 100 120 140 160 180 200 225 250 275 300 325 350 375 400 425 450 475 500 550 and 600 bases in length Figure 28 Each fragment is labeled with carboxy X rhodamine CXR and can be detected separately as a fourth color in the presence of PowerPlex 16 HS amplified material The ILS 600 is designed for use in each CE injection to increase precision in analyses when using the PowerPlex 16 HS System Protocols for preparation and use of this internal lane standard are provided in Section 5 10349TA Figure 28 Internal Lane Standard 600 An electropherogram showing the Internal Lane Standard 600 fragments 9 E Composition of Buffers and Solutions TE 4 buffer 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 final volume to 1 liter with deionized water TE 4 buffer with 20 g ml glycogen 1 21g Tris
87. llows co amplification and three color detection of sixteen loci fifteen STR loci and Amelogenin including Penta E D18S51 D21S11 TH01 D3S1358 FGA TPOX D8S1179 vWA Amelogenin Penta D CSF1PO D16S539 D7S820 D13S317 and D5S818 One primer for each of the Penta E D18S51 D21S11 TH01 and D3S1358 loci is labeled with fluorescein FL one primer for each of the FGA TPOX D8S1179 vWA and Amelogenin loci is labeled with carboxy tetramethylrhodamine TMR and one primer for each of the Penta D CSF1PO D16S539 D7S820 D13S317 and D5S818 loci is labeled with 6 carboxy 4 5 dichloro 2 7 dimethoxy fluorescein JOE All sixteen loci are amplified simultaneously in a single tube and analyzed in a single injection The PowerPlex 16 Monoplex System Penta E Fluorescein Cat DC6591 and PowerPlex 16 Monoplex System Penta D JOE Cat DC6651 are available to amplify the Penta E and Penta D loci respectively These monoplex systems allow amplification of a single locus to confirm results obtained with the PowerPlex 16 System or PowerPlex 16 HS System The PowerPlex 16 HS System is compatible with the ABI PRISM 310 3100 and 3100 Avant Genetic Analyzers and Applied Biosystems 3130 3130xl 3500 and 3500xL Genetic Analyzers The protocols presented in this manual were tested at Promega Amplification and detection instrumentation may vary You may need to optimize protocols including cycle number and injection conditions
88. loci not associated with a coding gene the first database entry or original literature description shall be used 2Amelogenin is not an STR but displays a 106 base X specific band and a 112 base Y specific band TMR carboxy tetramethylrhodamine FL fluorescein JOE 6 carboxy 4 5 dichloro 2 7 dimethoxyfluorescein NA not applicable Promega Corporation 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 TMD022 Revised 9 15 Table 3 The PowerPlex 16 HS System Allelic Ladder Information STR Locus Label Size Range of Allelic Ladder Components1 2 bases Repeat Numbers of Allelic Ladder Components3 Penta E FL 379 474 5 24 D18S51 FL 290 366 8 10 10 2 11 13 13 2 14 27 D21S11 FL 203 259 24 24 2 25 25 2 26 28 28 2 29 29 2 30 30 2 31 31 2 32 32 2 33 33 2 34 34 2 35 35 2 36 38 TH01 FL 156 195 4 9 9 3 10 11 13 3 D3S1358 FL 115 147 12 20 FGA TMR 322 444 16 18 18 2 19 19 2 20 20 2 21 21 2 22 22 2 23 23 2 24 24 2 25 25 2 26 30 31 2 43 2 44 2 45 2 46 2 TPOX TMR 262 290 6 13 D8S1179 TMR 203 247 7 18 vWA TMR 123 171 10 22 Amelogenin4 TMR 106 112 X Y Penta D JOE 376 449 2 2 3 2 7 17 CSF1PO JOE 321 357 6 15 D16S539 JOE 264 304 5 8 15 D7S820 JOE 215 247
89. ly 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 fluctuate Do not re freeze avoid multiple freeze thaw cycles as this may reduce activity Extreme variability in sample to sample peak heights There can be significant individual to individual variability in cell deposition onto buccal swabs This will appear as variability in peak heights between swab extracts The extraction process maximizes recovery of amplifiable DNA from buccal swabs but does not normalize the amount of DNA present If variability is extreme quantitate the DNA using a fluorescence based double stranded DNA quantitation method or qPCR based quantitation method The quantitation values can be used to normalize input template amounts to minimize variation in signal intensity 7 D GeneMapper ID X Software Symptoms Possible Causes and Comments Stutter peaks not filtered Stutter file was not imported into the Panel Manager when the panels and bins text files were imported Stutter distance was not defined in the Analysis Method Allele tab Be sure that the Use marker specific stutter ratio and distance if available box is checked Samples in the project not analyzed The Analysis Requirement Summary window is not active and there is an analysis requirement that has not been met Turn on Analysis Requirement Summary in the Options
90. lysis method dialog box will open 40 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD022 Revised 9 15 www promega com 6 D Creating a Databasing or Paternity Analysis Method with GeneMapper ID X Software Version 1 2 continued 4 In the Analysis Method Editor enter a descriptive name for the analysis method such as PowerPlex 16 HS_20 Filter 5 Choose a security group from the drop down menu 6 Select the Allele tab Figure 17 7 Select the bins text file that was imported in Section 6 A 8 We recommend the values shown in Figure 17 for proper filtering of stutter peaks when using the PowerPlex 16 HS System You may need to optimize these settings In house validation should be performed Note Ensure that the appropriate 20 filter is applied to this analysis method by entering 0 20 for the Global Cut off Value for Tetra and Penta repeats 9428TA Figure 17 The Allele tab with settings for using a 20 peak filter Promega Corporation 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 TMD022 Revised 9 15 9 Select the Peak Detector tab Figure 15 shows an example of settings used at Promega You may need to optimize these settings In house validation should be performed Notes 1 In the Ranges
91. m Tables 2 and 3 were selected because they satisfy the needs of several major standardization bodies throughout the world For example the United States Federal Bureau of Investigation FBI has selected 13 STR core loci for typing prior to searching or including submitting samples in CODIS Combined DNA Index System the U S national database of convicted offender profiles The PowerPlex 16 HS System amplifies all CODIS core loci in a single reaction The PowerPlex 16 HS System also contains two low stutter highly polymorphic pentanucleotide repeat loci Penta E and Penta D These additional loci add significantly to the discrimination power of the system making the PowerPlex 16 HS System a single amplification system with a power of exclusion sufficient to resolve paternity disputes definitively In addition the extremely low level of stutter seen with Penta E and Penta D makes them ideal loci to evaluate DNA mixtures often encountered in forensic casework Finally the Amelogenin locus is included in the PowerPlex 16 HS System to allow gender identification of each sample Table 4 lists the PowerPlex 16 HS System alleles revealed in commonly available standard DNA templates We have carefully selected STR loci and primers to avoid or minimize artifacts including those associated with Taq DNA polymerase such as repeat slippage and terminal nucleotide addition Repeat slippage 15 16 sometimes called n 4 bands stutter
92. maller than the allele Stutter product peak heights can be high if samples are overloaded See Section 6 J for additional information on stutter and artifacts Artifacts of STR amplification Amplification 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 the 30 minute extension step at 60 C after thermal cycling Section 4 Excessive amount of DNA Amplification of gt 1ng template can result in a higher number of stutter bands Use less template DNA or reduce the number of cycles in the amplification program by 2 4 cycles 10 20 or 10 18 cycling High background Load less amplification product or decrease injection time See Section 5 CE related artifacts spikes Minor voltage changes or urea crystals passing by the laser can cause spikes or unexpected peaks Spikes sometimes appear in one color but often are identified easily by their presence in more than one color Re inject samples to confirm CE related artifacts contaminants Contaminants in the water used with the instrument or to dilute the 10X genetic analyzer buffer may generate peaks in the blue and green dye colors Use autoclaved deionized water change vials and wash buffer reservoir Pull up or bleedthrough Pull up can occur when peak heights are too high or if a poor or incorrect matrix has been applied to the samples For the ABI PRISM 31
93. 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 first 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 then view the label edit table Marker header bars for some loci When an edit is made to a locus the quality flags and are gray 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 defined Promega Corporation 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 TMD022 Revised 9 15 Symptoms Possible Causes and Comments Alleles not called continued An insufficient number of ILS 600 fragments were defined Be sure to define at least two ILS 600 fragments smaller than the smallest sample peak or allelic ladder peak and at least two ILS 600 fragments larger than the largest sample peak or allelic ladder peak In this instance the allelic ladder would have failed the allelic ladder quality check Run was too short and larger peaks in ILS were not captured Not all ILS 600 peaks defined in the size standard were detected during the run Cre
94. mples and confirm that the PowerPlex 4 dye F spectral is set for F See instructions for instrument preparation in Section 5 Red bar appears during analysis of samples and the following error message appears when data are displayed Some selected sample s do not contain analysis data Those sample s will not be shown If none of the samples had matrices applied when run on the ABI PRISM 310 Genetic Analyzer no data will be displayed Apply a matrix file during analysis in the GeneMapper ID software and re analyze Promega Corporation 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 TMD022 Revised 9 15 Symptoms Possible Causes and Comments Error message after attempting to import panels and bins text files Unable to save panel data java SQLEException ORA 00001 unique constraint IFA CKP_NNN violated There was a conflict between different sets of panels and bins text files Check to be sure that the bins are installed properly If not delete all panels and bins text files and re import files in a different order Allelic ladder peaks labeled off ladder GeneMapper ID software was not used or microsatellite analysis settings were used instead of HID analysis settings GeneMapper software does not use the same algorithms as GeneMapper ID software and cannot correct for sizing differences using the all
95. need to be optimized in each laboratory for each sample type that is amplified 1 Place the MicroAmp plate in the thermal cycler Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 13 www promega com TMD022 Revised 9 15 2 Select and run the recommended protocol The preferred protocol for use with the GeneAmp PCR System 9700 thermal cycler is provided below Thermal Cycling Protocol1 96 C for 2 minutes then ramp 100 to 94 C for 30 seconds ramp 29 to 60 C for 30 seconds ramp 23 to 70 C for 45 seconds for 10 cycles then ramp 100 to 90 C for 30 seconds ramp 29 to 60 C for 30 seconds ramp 23 to 70 C for 45 seconds for 17 cycles then 60 C for 30 minutes 4 C soak 1When using the GeneAmp PCR System 9700 thermal cycler the ramp rates indicated in the cycling program must be set and the program must be run in 9600 ramp mode The ramp rates are set in the Ramp Rate Modification screen While viewing the cycling program navigate to the Ramp Rate Modification screen by selecting More then Modify On the Ramp Rate Modification screen the default rates for each step are 100 The rate under each hold step is the rate at which the temperature will change to that hold temperature Figure 2 shows the appropriate ramp rates for the GeneAmp PCR System 9700 thermal cycler The ramp mode is set after
96. nfo Windows NT operating systems column for each lane containing allelic ladder mix contains the word ladder The macro uses the word ladder to identify the sample file s containing allelic ladder Sample info can be added or modified after importing into the PowerTyper Macro Highlight the sample then select show dye lanes window in the Views menu 1 Transfer the PowerTyper 16 Macro Release 2 0 to a designated location on your computer hard drive 2 Open the Genotyper software then the PowerTyper 16 Macro Release 2 0 For questions about the Genotyper software refer to the Genotyper Analysis Software User s Manual 3 In the File menu select Import and import the GeneScan project or sample files to be analyzed Import the blue yellow green and red dye colors Note To select the dye colors to be imported select Set Preferences in the Edit menu 4 Double click on the Check ILS macro The macros are listed at the bottom left corner of the active window A plots window will be displayed to show the internal lane standard i e ILS 600 in the red dye color Scroll down to view and confirm that the internal lane standard fragment sizes are correct If necessary re analyze samples using the GeneScan software and redefine internal lane standard fragments Note The software uses one ladder sample to determine allele sizes The macro uses the first ladder sample imported for all
97. njection and sample electrophoresis 34 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD022 Revised 9 15 www promega com 6 Data Analysis 6 A PowerPlex 16 Panels Bins and Stutter Text Files with GeneMapper ID X Software Version 1 2 To facilitate analysis of data generated with the PowerPlex 16 HS System we have created panels and bins text files 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 Note The panels bins and stutter text files mentioned here are compatible with earlier versions of the GeneMapper ID X software Getting Started 1 To obtain the proper panels bins and stutter text files for the PowerPlex 16 HS System go to www promega com resources tools genemapper id software panels and bin sets 2 Enter your contact information and select GeneMapper ID X Select Submit 3 Save the PowerPlex_16_Panels_IDX_vX x txt PowerPlex_16_Bins_IDX_vX x txt and PowerPlex_16_ Stutter_IDX_vX x txt files where X x refers to the most recent version of the panels bins and stutter text files to a known location on your computer Importing Panels Bins and Stutter Text Files 1 Open the GeneMapper ID
98. nt Summary Allelic Ladder Analysis Summary and Analysis Summary windows after quality review by the software Ensure that all requirements are met as each window appears If you do not have the Analysis Requirement Summary window activated you may need to do additional manual troubleshooting 10 If all analysis requirements are met the Save Project window will open Figure 16 11 Enter the project name 12 Choose the applicable security group from the drop down menu then select OK When the analysis is finished 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 Navigate to the Genotype tab or Samples tab To assist the review of any low quality samples use the default Data Interpretation plot settings and review the contents in the Quality Value Details table The values displayed in the Analysis Method Peak Quality and SQ amp GQ Settings tabs are defaults and will affect the quality values displayed in the plot settings We recommend that you modify the values in these tabs to fit your laboratory s data analysis protocols 6 E PowerPlex Panels and Bins Text Files with GeneMapper ID Software Version 3 2 To facilitate analysis of data generated with the PowerPlex 16 HS System we have created panels and bins text files to allow automatic assignment of genotypes using GeneMap
99. nued 4 B Direct Amplification of DNA from Storage Card Punches Materials to Be Supplied by the User GeneAmp PCR System 9700 thermal cycler Applied Biosystems microcentrifuge MicroAmp optical 96 well reaction plate Applied Biosystems aerosol resistant pipette tips 5X AmpSolution Reagent Cat DM1231 also supplied with the PunchSolution Kit PunchSolution Kit Cat DC9271 for nonFTA punches 1 2mm Harris Micro Punch or equivalent manual punch and cutting mat This section contains a protocol for direct amplification of DNA from storage card punches using the PowerPlex 16 HS 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 amplification reaction Note You will need to optimize and validate the number of storage card punches per reaction in your laboratory FTA based sample types include Buccal cells collected on FTA cards with Whatman EasiCollect or Fitzco Sampact devices one or two punch per 25 l amplification reaction Buccal cells collected with sterile swabs transferred to FTA or Indicating FTA cards one or two punch per 25 l amplification reaction Liquid blood from collection or storage Vacutainer tubes or finger sticks spotted onto FTA cards one punch per 25 l amplification reaction NonFTA sample types include one
100. number of storage card punches 4 C Direct Amplification of DNA from Swabs Materials to Be Supplied by the User GeneAmp PCR System 9700 thermal cycler Applied Biosystems microcentrifuge MicroAmp optical 96 well reaction plate Applied Biosystems aerosol resistant pipette tips SwabSolution Kit Cat DC8271 This section contains a protocol for amplifying swab extracts using the PowerPlex 16 HS System and GeneAmp PCR System 9700 thermal cycler Pretreat cotton or OmniSwabs GE Healthcare swabs with the SwabSolution Kit Cat DC8271 as described in the SwabSolution Kit Technical Manual TMD037 to generate a swab extract Amplification Setup 1 Thaw the PowerPlex HS 5X Master Mix and PowerPlex 16 HS 10X Primer Pair Mix completely Note Centrifuge tubes briefly 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 amplification mix for all samples It also ensures that each reaction contains the same PCR amplification mix
101. o the well before adding the punch may help alleviate static problems Amplification Setup 1 Thaw the PowerPlex HS 5X Master Mix and PowerPlex 16 HS 10X Primer Pair Mix completely Note Centrifuge tubes briefly 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 amplification mix for all samples It also ensures that each reaction contains the same PCR amplification mix 3 Use a clean MicroAmp plate for reaction assembly and label appropriately 4 Add the final volume of each reagent listed in Table 2 to a sterile tube Table 2 PCR Amplification Mix for Direct Amplification of DNA from Storage Card Punches PCR Amplification Mix Component1 Volume Per Reaction Number of Reactions Final Volume Water Amplification Grade 12 5 l PowerPlex HS 5X Master Mix 5 0 l PowerPlex 16 HS 10X Primer Pair Mix 2 5 l 5X AmpSolution Reagent 5 0 l total reaction volume 25 l 1Add Water Amplification Grade to t
102. ods 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 TMD022 Revised 9 15 Symptoms Possible Causes and Comments Faint or absent allele peaks continued Active protease from the SwabSolution Reagent carried over into the amplification reaction Ensure that the heat block is heating to 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 protease inactivation Do not use an incubator set at 70 C to incubate tubes or plates Heat transfer is inefficient and will result in poor performance Only use a heat block to maintain efficient heat transfer We have tested 60 minute incubation times and observed no difference in performance compared to a 30 minute incubation Make sure that the PCR amplification mix also contained AmpSolution Reagent Omission of AmpSolution Reagent from amplification reactions will result in amplification failure Faint or absent peaks for the positive control reaction If the positive control reaction failed to amplify check to make 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 profile We recommend 5ng of 2800M Control DNA per 25 l amplification reaction
103. ollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 77 www promega com TMD022 Revised 9 15 3 There can be variation between instruments regarding the relative fluorescence levels detected using the same sample Furthermore different instruments vary in the relative efficiency of color detection affecting the dye color to dye color balance Sample Analysis Using the Genotyper Software and PowerTyper 16 Macro To facilitate analysis of data generated with the PowerPlex 16 HS System we have created a file to allow automatic assignment of genotypes using the Genotyper software After samples are amplified detected using the ABI PRISM 310 or 3100 Genetic Analyzer using Data Collection Software Version 1 0 1 or 1 1 and analyzed using the GeneScan software sample files can be imported into the Genotyper program and analyzed using the PowerTyper 16 Macro Release 2 0 The PowerTyper 16 Macro Release 2 0 is available for download from the Promega web site at www promega com resources tools powertyper macros The PowerTyper 16 Macro Release 2 0 is used in conjunction with Macintosh Genotyper software version 2 5 and Windows NT Genotyper software version 3 6 or later The Genotyper software must be installed on your computer before the PowerTyper 16 Macro Release 2 0 can be used Be certain the Sample Info Macintosh computers or Color I
104. on This can be accomplished with post PCR cleanup or desalting lower conductivity formamide or reduced amounts of ILS 600 In house validation should be performed for any of these methods Insufficient enzyme activity Vortex the PowerPlex HS 5X Master Mix before use and use the recommended amount Incorrect amplification program Confirm the amplification program The PowerPlex HS 5X Master Mix was not vortexed well before use Vortex the 5X Master Mix for 5 10 seconds before dispensing into reaction tubes or plates An air bubble formed at the bottom of the reaction tube Use a pipette to remove the air bubble or centrifuge the reactions briefly before thermal cycling High salt concentration or altered pH If the DNA template is stored in TE buffer that is not pH 8 0 or contains a higher EDTA concentration the DNA volume should not exceed 20 of the total reaction volume Carryover of K Na Mg2 or EDTA from the DNA sample can negatively affect PCR A change in pH also may affect PCR Store DNA in TE 4 buffer 10mM Tris HCl pH 8 0 0 1mM EDTA or nuclease free water Thermal cycler plate or tube problems Review the thermal cycling protocols 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 10X PowerPlex 16 HS Primer Pair for 15 seconds before us
105. on of this control DNA by other methods such as qPCR may result in a different value Prepare a fresh DNA dilution for each set of amplifications Do not store diluted DNA e g 0 25ng l or less Promega Corporation 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 TMD022 Revised 9 15 4 A Amplification of Extracted DNA Materials to Be Supplied by the User GeneAmp PCR System 9600 or 9700 thermal cycler Applied Biosystems microcentrifuge 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 the protocol detailed below With gt 1ng of DNA preferential amplification of smaller loci can occur Expect to see high peak heights at the smaller loci and lower peak heights at the larger loci if more than the recommended amount of template is used Reduce the amount of template DNA or the number of cycles to correct this Amplification Setup 1 Thaw the PowerPlex HS 5X Master Mix and PowerPlex 16 HS 10X Primer Pair Mix completely Note Centrifuge tubes briefly 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
106. or loading volume for each laboratory instrument In house validation should be performed 7 E GeneMapper ID Software 62 7 F PowerTyper 16 Macro 65 8 References 67 9 Appendix 69 9 A Advantages of Using the Loci in the PowerPlex 16 HS System 69 9 B Power of Discrimination 73 9 C Sample Analysis Using the GeneScan and Genotyper Software 74 9 D The Internal Lane Standard 600 80 9 E Composition of Buffers and Solutions 80 9 F DNA Extraction and Quantitation Methods and Automation Support
107. orescein dye channel One or more extra peaks that are not directly related to amplification may be observed in the D3S1358 TH01 D21S11 and Penta E region of the fluorescein channel D13S317 and D16S539 region of the JOE channel and vWA region of the TMR channel These extra peaks occur when the amplified peaks are particularly intense high signal intensity or template amount formamide polymer or capillary was of poor quality or denaturation was ineffective See Section 7 for more information on how to minimize these artifacts Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 53 www promega com TMD022 Revised 9 15 7 Troubleshooting For questions not addressed here please contact your local Promega Branch Office or Distributor Contact information available at www promega com E mail genetic promega com 7 A Amplification and Fragment Detection Symptoms Possible Causes and Comments Faint or absent allele peaks Impure template DNA Because of the small amount of template used this is rarely a problem Depending on the DNA extraction procedure used and sample source inhibitors might be present in the DNA sample Insufficient template Use the recommended amount of template DNA Insufficient template Low copy number LCN analysis using capillary electrophoresis may benefit from reducing competing charged particles during injecti
108. owerPlex 16 HS System in Various Populations African American Caucasian American Hispanic American Asian American Matching Probability 1 in 1 41 1018 1 in 1 83 1017 1 in 2 93 1017 1 in 3 74 1017 Paternity Index 2 510 000 1 520 000 522 000 4 110 000 Power of Exclusion 0 9999996 0 9999994 0 9999983 0 9999998 74 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD022 Revised 9 15 www promega com 9 C Sample Analysis Using the GeneScan and Genotyper Software Sample Analysis Using the GeneScan Software and Windows Operating Systems 1 Analyze data using the GeneScan software 2 Review the raw data for one or more sample runs Highlight the sample file name then in the Sample menu select raw data Move the cursor so that the crosshair is on the baseline to the right of the large primer peak before the first internal lane standard peak red Use the X value number shown at the bottom left of the window for the start position in the analysis parameters 3 The recommended analysis parameters are shown in Figure 27 5684TA Figure 27 The Analysis Parameters window The start point of the analysis range which will vary is defined in Step 2 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 75
109. per ID Software Version 3 2 continued 6 Select the Allele tab Figure 20 7 Select the bins text file that was imported in Section 6 E 8 Ensure that the Use marker specific stutter ratio if available box is checked 5724TA Figure 20 The GeneMapper ID Allele tab 9 Enter the values shown in Figure 20 for proper filtering of stutter peaks when using the PowerPlex 16 HS System For an explanation of the proper usage and effects of these settings refer to the Applied Biosystems user bulletin titled Installation Procedures and New Features for GeneMapper ID Software 3 2 Note Some of these settings have been optimized and are different from the recommended settings in the user bulletin Promega Corporation 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 TMD022 Revised 9 15 10 Select the Peak Detector tab We recommend the settings shown in Figure 21 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 first defined 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 ar
110. per ID software version 3 2 We recommend that users of GeneMapper ID software version 3 2 complete the Applied Biosystems GeneMapper ID Software Human Identification Analysis Tutorial to familiarize themselves with proper operation of the software For GeneMapper ID software version 3 1 users we recommend upgrading to version 3 2 For analysis using GeneMapper ID software version 3 2 you will need the proper panels and bins text files PowerPlex_16_Panels_vX x txt and PowerPlex_16_Bins_vX x txt files where X x refers to the most recent version of the panels and bins text files Promega Corporation 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 TMD022 Revised 9 15 Getting Started 1 To obtain the panels and bins text files for the PowerPlex 16 HS 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 PowerPlex_16_Panels_vX x txt and PowerPlex_16_Bins_vX x txt files to a known location on your computer Importing Panels and Bins Text Files These instructions loosely follow the Applied Biosystems GeneMapper ID software tutorial pages 1 4 1 Open the GeneMapper ID software version 3 2 2 Select Tools then
111. plification 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 amplification control pipet Water Amplification Grade or TE 4 buffer instead of swab extract into a reaction well containing PCR amplification 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 the plate Optional Briefly centrifuge the plate to bring contents to the bottom of the wells and remove any air bubbles 16 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD022 Revised 9 15 www promega com 4 C Direct Amplification of DNA from Swabs continued Thermal Cycling Amplification 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 28 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 amplified see below 1 Place the MicroAmp plate in the thermal cycler 2 Select and run the recommended protocol
112. rd punch with 10 l of PunchSolution Reagent and using one punch per 25 l amplification reaction Use of a smaller amplification reaction volume may compromise performance if using 10 l of PunchSolution Reagent Reducing the PunchSolution Reagent volume may improve results when using a reduced amplification reaction volume Laboratory optimization and validation are required Extreme variability in sample to sample peak heights There can be significant individual to individual variability in the deposition of cells onto a punch resulting in peak height variability between samples The PunchSolution Kit increases the recovery of amplifiable DNA from samples but does not normalize the amount of DNA present 7 C Amplification of DNA from Swabs The following information is specific to amplification of DNA from swabs For information about general amplification and detection see Section 7 A Symptoms Possible 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 fluctuate Do not refreeze avoid multiple freeze thaw cycles as this may reduce activity Promega Corporation 2800 Wo
113. reactions DC6700 Differex System 50 samples DC6801 200 samples DC6800 Tissue and Hair Extraction Kit for use with DNA IQ 100 reactions DC6740 Maxwell 16 Forensic Instrument 1 each AS3060 DNA IQ Reference Sample Kit for Maxwell 16 48 preps AS1040 DNA IQ Casework Pro Kit for Maxwell 16 48 preps AS1240 Plexor HY System 200 reactions DC1001 800 reactions DC1000 Slicprep 96 Device 10 pack V1391 Not for Medical Diagnostic Use 10 Summary of Changes The following change was made to the 9 15 revision of this document 1 The document design was updated 84 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD022 Revised 9 15 www promega com a U S Pat No 6 238 863 Chinese Pat No ZL99802696 4 European Pat No 1058727 Japanese Pat No 4494630 and other patents pending b Australian Pat No 724531 Canadian Pat No 2 251 793 Korean Pat No 290332 Singapore Pat No 57050 Japanese Pat Nos 3602142 and 4034293 Chinese Pat Nos ZL99813729 4 and ZL97194967 0 European Pat No 0960207 and other patents pending c Allele sequences for one or more of the loci vWA FGA D8S1179 D21S11 and D18S51 in allelic ladder mixtures is licensed under U S Pat Nos 7 087 380 and 7 645 580 Australia Pat No 2003200444 and corresponding patent claims outside the US 2009 2015
114. s occurs at a heterozygous locus it is sometimes possible to see two shadow peaks that differ in size from one another by approximately the same distance as the single stranded alleles Artifacts of STR amplification Direct amplification of gt 20ng of template can result in a higher number of artifact peaks Use the recommended punch size and number of punches Optimize the cycle number Do not reduce the reaction volume below 25 l See Section 6 L for additional information on stutter and artifacts Artifacts of STR amplification Amplification 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 30 minute extension step at 60 C after thermal cycling Section 4 Peak height imbalance Excessive amount of DNA Amplification of gt 20ng of template can result in an imbal ance with smaller loci showing more product than larger loci Use one or two 1 2mm punches from a storage card containing a buccal sample or one 1 2mm punch from a storage card containing whole blood Follow the manufacturer s recommendations when depositing sample onto the storage card Decrease number of cycles The reaction volume was too low This system is optimized for a final reaction volume of 25 l to overcome inhibitors present in storage cards and PunchSolution Reagent Decreasing the reaction volume can result in suboptimal performance Ampl
115. s section contains protocols for use of the PowerPlex 16 HS System with the GeneAmp PCR system 9600 and 9700 thermal cyclers For information on other thermal cyclers contact Promega Technical Services by e mail genetic promega com Amplification and detection instrumentation may vary You may need to optimize protocols including cycle number and injection conditions or loading volume for each laboratory instrument Testing at Promega shows that 10 22 cycles work well for 0 5ng of purified DNA templates For higher amounts of input DNA i e FTA paper or to decrease sensitivity fewer cycles such as 10 16 10 18 or 10 20 should be evaluated In house validation should be performed 1 Place reaction tubes or MicroAmp plate in the thermal cycler 2 Select and run the recommended protocol The preferred protocols for use with the GeneAmp PCR System 9600 and 9700 thermal cyclers are provided below 3 After completion of the thermal cycling protocol store amplified samples at 20 C in a light protected box Note Long term storage of amplified samples at 4 C or higher may produce artifacts Protocol for the GeneAmp PCR System 9600 Thermal Cycler Protocol for the GeneAmp PCR System 9700 Thermal Cycler1 96 C for 2 minutes then 96 C for 2 minutes then 94 C for 30 seconds ramp 68 seconds to 60 C hold for 30 seconds ramp 50 seconds to 70 C hold for 45 seconds for 10 cycles then ramp 100 to 94 C for
116. section select Full Range in the Analysis drop down menu and Partial Sizes in the Sizing drop down menu 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 first defined 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 on the ABI PRISM 310 3100 and 3100 Avant Genetic Analyzers and Applied Biosystems 3130 and 3130xl Genetic Analyzers but should be determined by individual laboratories 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 peak amplitude thresholds from internal validation studies 3 The normalization box can be checked regardless of whether normalization was or was not applied during data collection 10 Select the Peak Quality tab You may change the settings for peak quality Note For Steps 11 and 12 see the GeneMapper ID X user s manual for more information 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
117. standard was not correctly defined or size peaks were missing Redefine size standard to include only peaks present in your sample Terminating analysis early or using short run times will cause larger ladder peaks to be missing This will cause your sizing quality to be flagged as red and no allele sizes will be called Error message Both the Bin Set used in the Analysis Method and the Panel must belong to the same Chemistry Kit The bins text file assigned to the analysis method was deleted In the GeneMapper Manager select the Analysis Methods tab and open the analysis method of interest Select the Allele tab and select an appropriate bins text file The wrong bins text file was chosen in the analysis method Allele tab Be sure to choose the appropriate bins text file as shown in Figure 20 Significantly raised baseline Poor spectral calibration for the ABI PRISM 3100 and 3100 Avant Genetic Analyzers and Applied Biosystems 3130 3130xl 3500 and 3500xL Genetic Analyzers Perform a new spectral calibration and re run the samples Poor matrix for the ABI PRISM 310 Genetic Analyzer Re run and optimize the matrix Use of Classic mode analysis method Use of Classic mode analysis on samples can result in baselines with more noise than those analyzed using the Basic or Advanced mode analysis method Advanced mode analysis methods and size standards are recommended Incorrect spectral was active Re run sa
118. ternal lane standard used in the loading cocktail can be increased or decreased to adjust the intensity of the size standard peaks If peak heights are too low we recommend altering the formamide internal lane standard mix to contain 1 0 l of ILS 600 and 9 0 l of Hi Di formamide If peak heights are too high we recommend altering the loading cocktail to contain 0 25 l of ILS 600 and 9 75 l of formamide 2 Vortex for 10 15 seconds to mix 3 Pipet 10 l of formamide internal lane standard mix into each well 4 Add 1 l of amplified sample or 1 l of PowerPlex 16 HS Allelic Ladder Mix 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 increased or decreased To modify the injection time or injection voltage 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 amplification reactions or reduce the number of cycles in the amplification program by 2 4 cycles to achieve the desired signal intensity 5 Centrifuge plate briefly to remove air bubbles from the wells 6 Denature samples at 95 C for 3 minutes then immediately chill on crushed ice or in an ice water bath for 3 minutes Denature samples just prior to loading the instrument Promega Corporation 2800 Woods
119. that was imported in Section 6 E 7 In the Size Standard column select the size standard that was created in Section 6 F 8 If analyzing data from an ABI PRISM 310 Genetic Analyzer ensure that the appropriate matrix file is selected in the Matrix column 9 Select Analyze green arrow button to start data analysis 6 H Creating a Databasing or Paternity Analysis Method 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 In the Analysis Method Editor enter a descriptive name for the analysis method such as PowerPlex16_20 filter 6 Select the Allele tab Figure 22 7 Select the bins text file that was imported in Section 6 E 8 Ensure that the Use marker specific stutter ratio if available box is checked Promega Corporation 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 TMD022 Revised 9 15 9 Enter the values shown in Figure 22 for proper filtering of peaks when using the PowerPlex 16 HS System For an explanation of the proper usage and effect of these settings refer to the Appli
120. the location of the run files Highlight desired files 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 5 In the Analysis Method column select the analysis method created above 6 In the Panel column select the panels text file that was imported in Section 6 A 7 In the Size Standard column select the size standard that was created in Section 6 B 8 If analyzing data from an ABI PRISM 310 Genetic Analyzer ensure that the appropriate matrix file is selected in the Matrix column 9 Select Analyze green arrow button to start data analysis Note By default the software displays the Analysis Requirement Summary Allelic Ladder Analysis Summary and Analysis Summary windows after quality review by the software Ensure that all requirements are met as each window appears If you do not have the Analysis Requirement Summary window activated you may need to do additional manual troubleshooting Promega Corporation 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 TMD022 Revised 9 15
121. the relative efficiency of color detection affecting the dye color to dye color balance Sample Analysis Using the GeneScan Software and Macintosh Operating Systems 1 Analyze data using the GeneScan software 2 Review the raw data for one or more sample runs Highlight the sample file name then in the Sample menu select raw data Move the cursor so that the crosshair is on the baseline to the right of the large primer peak before the first internal lane standard peak red Use the X value number shown at the bottom left of the window for the start position in the analysis parameters 76 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD022 Revised 9 15 www promega com Sample Analysis Using the GeneScan Software and Macintosh Operating Systems continued 3 The recommended analysis parameters are Analysis Range Start Defined in Step 2 Stop 10 000 Data Processing Baseline Checked Multicomponent Checked Smooth Options Light1 Peak Detection Peak Amplitude Thresholds2 B Y G R Min Peak Half Width 2pt Size Call Range Min 60 Max 600 Size Calling Method Local Southern Method Split Peak Correction None 1Smooth options should be determined by individual laboratories Occasionally the TH01 alleles 9 3 and 10 will not be distinguished using heavy smoothing 2The peak amplitud
122. thod such as PowerPlex 16 HS 5 Choose a security group from the drop down menu 6 Select the Allele tab Figure 14 9426TA Figure 14 The GeneMapper ID X Allele tab Promega Corporation 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 TMD022 Revised 9 15 7 Select the bins text file that was imported in Section 6 A 8 Ensure that the Use marker specific stutter ratio and distance if available box is checked 9 We recommend the values shown in Figure 14 for proper filtering of stutter peaks when using the PowerPlex 16 HS System You may need to optimize these settings In house validation should be performed 10 Select the Peak Detector tab Figure 15 shows an example of settings used at Promega You may need to optimize these settings In house validation should be performed Notes 1 In the Ranges section select Full Range in the Analysis drop down menu and Partial Sizes in the Sizing drop down menu If 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 first defined internal lane standard peak to help ensure proper sizing of the internal lane standard 9427TA Figure 15 The GeneMapper ID X Peak Detector tab 38 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA To
123. tion 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 TMD022 Revised 9 15 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 workflow 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 different cycle number 27 28 and 29 cycles Note This recommendation is for 2 l of swab extract Additional cycle number testing may be required 4 Following amplification use your laboratory s validated separation and detection protocols to determine the optimal cycle number for the sample type 5 Instrument Setup and Sample Preparation 5 A Detection of Amplified Fragments Using the Applied Biosystems 3500 or 3500xL Genetic Analyzer Materials to Be Supplied by the User 95 C dry heating block water bath or thermal cycler crushed ice or ice water bath centrifuge compatible with 96 well plates aerosol resistant pipette tips 3500 3500xL capillary array 36cm 96 well retainer amp base set standard Applied
124. trix Standards 310 Cat DG4640 and the ABI PRISM 3100 and 3100 Avant Genetic Analyzers and Applied Biosystems 3130 3130xl 3500 and 3500xL Genetic Analyzers PowerPlex Matrix Standards 3100 3130 Cat DG4650 See Section 9 G for ordering information 3 Before You Begin 3 A Precautions The application of PCR based typing for forensic or paternity casework requires validation studies and quality control measures that are not contained in this manual 10 11 Guidelines for the validation process are published in the Internal Validation of STR Systems Reference Manual 12 The quality of purified DNA small changes in buffers ionic strength primer concentrations choice of thermal cycler and thermal cycling conditions can affect PCR success We suggest strict adherence to recommended procedures for amplification and fluorescence detection Additional research and validation are required if any modifications 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 amplification reactions and analyzing amplification products Reagents and materials used prior to amplification PowerPlex HS 5X Master Mix PowerPlex 16 HS 10X Primer Pair Mix 2800M Control DNA and Water Amplification Grade are provided in a separate box and should be stored sep
125. ty 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 Prepare a loading cocktail by combining Internal Lane Standard 600 ILS 600 and Hi Di formamide as follows 1 0 l ILS 600 samples 24 0 l Hi Di formamide samples Note The volume of internal lane standard used in the loading cocktail can be increased or decreased to adjust the intensity of the size standard peaks The optimal peak height for the 100 base fragment of the internal lane standard is 500 1 000RFU If peak heights are too high we recommend altering the loading cocktail to contain 0 5 l of ILS 600 and 24 5 l of Hi Di formamide 2 Vortex for 10 15 seconds to mix 3 Combine 25 0 l of prepared loading cocktail and 1 0 l of amplified sample or 1 0 l of PowerPlex 16 HS Allelic Ladder Mix Note Instrument detection limits vary therefore injection time injection voltage or the amount of product mixed with loading cocktail may need to be increased or decreased Modify the injection time or voltage in the injection list If peak heights still are hig
126. unch may inhibit amplification of 2800M Control DNA Optimize the amount of 2800M Control DNA for your thermal cycling conditions and laboratory preferences Promega Corporation 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 TMD022 Revised 9 15 Symptoms Possible Causes and Comments Extra peaks visible in one or all color channels Punch was contaminated Take punches from blank paper samples between samples Amplification of processed punches with high amounts of DNA can result in artifact peaks due to overamplification resulting in saturating signal on the CE instrument We recommend one or two 1 2mm punches per 25 l reaction Use of a larger punch size or a smaller reaction volume may result in overamplification and signal saturation If the signal is saturated repeat the amplification with a smaller punch a larger reaction volume or reduced cycle number Amplification 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 difficult 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 If thi
127. ysis of sample data is desired refer to the instrument user s manual for instructions 4 In the GeneMapper plate record enter sample names in the appropriate cells Scroll to the right In the Results Group 1 column select the desired results group In the Instrument Protocol 1 column select the protocol you created in Step 2 Be sure this information is present for each row that contains a sample name Select OK Note To create a new results group select New in the drop down menu in the Results Group column Select the General tab and enter a name Select the Analysis tab and select GeneMapper Generic in the Analysis type drop down list 5 Place samples in the instrument and close the instrument doors 6 In the spectral viewer confirm that dye set F is active and set the correct active spectral calibration for dye set F 7 In the run scheduler locate the plate record that you just created in Steps 3 and 4 and click once on the name to highlight it 8 Once the plate record is highlighted click the plate graphic that corresponds to the plate on the autosampler that contains your amplified samples 9 When the plate record is linked to the plate the plate graphic will change from yellow to green and the green Run Instrument arrow becomes enabled 10 Click on the green Run Instrument arrow on the toolbar to start the sample run 11 Monitor electrophoresis by observing the run view array or

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PowerPlex® 16 HS Technical Manual