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1. 5 B Direct Amplification of DNA from Storage Card Punches 8 C Direct Amplification of DNA from Swabs 11 5 Instrument Setup and Sample Preparation 14 A Detection of Amplified Fragments Using the Applied Biosystems 3500 or 3500xL Genetic Analyzer 14 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 25 6 Data Analysis 27 A PowerPlex 21 Panels Bins and Stutter Text Files with GeneMapper ID X Software Version 1 2 27 B Creating a Size Standard with GeneMapper ID X Software Version 1 2 28 C Importing the CC5 ILS 500 IDX Size Standard into GeneMapper ID X Software Version 1 2 30 D Creating a Casework Analysis Method with GeneMapper ID X Software Version 1 2
2. 30 E Creating a Databasing or Paternity Analysis Method with GeneMapper ID X Software Version 1 2 34 F PowerPlex 21 Panels and Bins Text Files with GeneMapper ID Software Version 3 2 37 G Creating a Size Standard with GeneMapper ID Software Version 3 2 38 H Importing the CC5 ILS 500 Size Standard into GeneMapper ID Software Version 3 2 40 I Creating a Casework Analysis Method with GeneMapper ID Software Version 3 2 40 J Creating a Databasing or Paternity Analysis Method with GeneMapper ID Software Version 3 2 43 K Controls 45 L Results 45 Page 1 7 Troubleshooting 48 A Amplification and Fragment Detection 48
3. 3 Select New and a new analysis method dialog box will open 4 Select HID and select OK Note If you do not see the HID option you do not have the GeneMapper ID software Contact Applied Biosystems 5 Enter a descriptive name for the analysis method such as PowerPlex21 6 Select the Allele tab Figure 19 7 Select the bins text file that was imported in Section 6 F 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 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD034 Printed in USA Revised 7 14 Page 40 9 Enter the values shown in Figure 19 for proper filtering of stutter peaks when using the PowerPlex 21 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 Page 41 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD034 Revised 7 14 9778TA Figure 19 The GeneMapper ID Allele tab 6 I Creat
4. 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 instrument user s manual for instructions on cleaning installing the capillary array performing a spatial calibration and adding polymer Analyze samples as described in the user s manual for the ABI PRISM 3100 or 3100 Avant Genetic Analyzer with Data Collection Software Version 2 0 and the Applied Biosystems 3130 or 3130xl Genetic Analyzer with Data Collection Software Version 3 0 with the following exceptions 1 In the Module Manager select 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 the injection voltage is 3kV and the run time is 1 500 seconds Give a descriptive name to your run module and select OK Note Instrument sensitivities can vary The injection time and voltage may be adjusted in the Module Manager A suggested range for the injection time is 3 22 seconds and for the injection voltage is 1 3kV 2 In the Protocol Manager select New Type a name for your protocol Select Regular in the Type drop down list and select the run module you created in the previous step in the R
5. Fax 608 277 2516 www promega com Printed in USA Part TMD034 Revised 7 14 5 Place samples in the instrument and close the instrument doors 6 In the spectral viewer select dye set G5 and confirm that the active dye set is the file generated for the PowerPlex 5 dye chemistry It is critical to select the correct G5 spectral for the PowerPlex 5 dye chemistry If the PowerPlex 5 dye chemistry is not the active dye set locate the PowerPlex 5 dye spectral in the List of Calibrations for Dye Set G5 and select Set 7 In the run scheduler locate the plate record that you just created in Steps 3 and 4 and click once on the name to highlight it 8 Once the plate record is highlighted 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 capillaries viewer window in the data collection software Each injection will take approximately 40 minutes 6 Data Analysis 6 A PowerPlex 21 Panels Bins and Stutter Text Files with GeneMapper ID X Software Version 1 2 To facilitate analysis of data generated with the PowerPlex 21 System we have
6. Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD034 Printed in USA Revised 7 14 Figure 22 The PowerPlex 21 System A single source template DNA 0 5ng was amplified using the PowerPlex 21 System Amplification products were mixed with CC5 Internal Lane Standard 500 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 Amelogenin D3S1358 D1S1656 D6S1043 D13S317 and Penta E Panel B An electropherogram showing the peaks of the JOE labeled loci D16S539 D18S51 D2S1338 CSF1PO and Penta D Panel C An electropherogram showing the peaks of the TMR ET labeled loci TH01 vWA D21S11 D7S820 D5S818 and TPOX Panel D An electropherogram showing the peaks of the CXR ET labeled loci D8S1179 D12S391 D19S433 and FGA Panel E An electropherogram showing the 60bp to 500bp fragments of the CC5 Internal Lane Standard 500 A B C D E 10247TA Page 47 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD034 Revised 7 14 10345TA A B C D Figure 23 The PowerPlex 21 Allelic Ladder Mix The PowerPlex 21 Allelic Ladder Mix was an
7. 5 A Detection of Amplified Fragments Using the Applied Biosystems 3500 or 3500xL Genetic Analyzer continued 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 18 In the Run Information window Figure 11 assign a Run Name Select Start Run not shown Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD034 Printed in USA Revised 7 14 Page 24 Figure 11 Assigning a run name 9256TA Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD034 Revised 7 14 5 B Detection of Amplified Fragments Using the ABI PRISM 3100 or 3100 Avant Genetic Analyzer with Data Collection So
8. B Direct Amplification of DNA From Storage Card Punches 51 C Direct Amplification of DNA From Swabs 53 D GeneMapper ID X Software 55 E GeneMapper ID Software 57 8 References 59 9 Appendix 60 A Advantages of Using the Loci in the PowerPlex 21 System 60 B DNA Extraction and Quantitation Methods and Automation Support 64 C The CC5 Internal Lane Standard 500 65 D Composition of Buffers and Solutions 65 E Related Products 66 F Summary of Changes 67 1 Description STR short tandem repeat loci consist of s
9. Max for the ramp speed and enter the reaction volume 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 PunchSolution Kit Cat DC9271 for nonFTA card 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 21 System and GeneAmp PCR System 9700 thermal cycler 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 punches per 25 l amplification reaction Buccal cells collected with sterile swabs transferred to FTA or Indicating FTA cards one or two punches 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 Buccal samples on Bode Buccal DNA Collector devices one punch per 25 l amplification reaction Blood and buccal samples on nonFTA card punches e g S a
10. Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD034 Printed in USA Revised 7 141 Page 32 8259TA Figure 14 The GeneMapper ID X Peak Detector tab Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD034 Revised 7 14 those for the other dyes Therefore the threshold for the orange dye may be lower than that for the other dyes 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 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
11. Quantification 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 4 A Amplification of Extracted DNA Materials to Be Supplied by the User GeneAmp PCR System 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 Amplification Setup 1 Thaw the PowerPlex 21 5X Master Mix and PowerPlex 21 5X 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 5X Primer Pair Mix or 5X Master Mix after vortexing as this may cause the reagents to be concentrated at the bottom of the tube Page 5 4 A Amplification of Extracted DNA continued 2 Determine the number of reactions to be set up This should include positive and negative control reactions Add 1 or 2 reactions to this number to compensate for pipetting error While this approach does 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 amplificatio
12. TE 4 buffer 10mM Tris HCl pH 8 0 0 1mM EDTA or TE 4 buffer with 20 g ml glycogen 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 PCR amplification efficiency and quality can be greatly altered by changes in pH due to added Tris HCl available magnesium concentration due to chelation by EDTA or other PCR inhibitors which may be present at low concentrations depending on the source of the template DNA and the extraction procedure used 3Apparent DNA concentrations can differ depending on the DNA quantification method used 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 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 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 or close the tubes Optional Briefly centrifuge the plate to bring contents to the bottom of the wells and remove any air
13. WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD034 Revised 7 14 Page 9 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 15 l PowerPlex 21 5X Master Mix 5 0 l PowerPlex 21 5X Primer Pair Mix 5 0 l total reaction volume 25 l 1Add Water Amplification Grade to the tube first then add PowerPlex 21 5X Master Mix and PowerPlex 21 5X Primer Pair Mix For FTA card punches the template DNA will be added at Step 6 4 B Direct Amplification of DNA from Storage Card Punches continued 7 For the positive amplification control add 1 l of 2800M Control DNA 10ng l 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 depending on thermal cycling conditions and laboratory preferences 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
14. freeze thaw cycles as this may reduce activity Extreme variability in sample There can be significant individual to individual variability in to sample peak heights cell deposition onto buccal swabs This will appear as variability in peak heights between swab extracts The extraction process maximizes recovery of 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 Causes and Comments Stutter peaks not filtered Stutter text 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 Samples in the project not analyzed The Analysis Requirement Summary window was not active and there was an analysis requirement that was not met Turn on Analysis Requirement Summary in the Options menu and correct the necessary analysis requirements to continue analysis Edits in label edit viewer cannot To view edits made to a project the project first must be be viewed 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 ed
15. 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 downloaded in the Getting Started section above Select the file then Import 6 In the navigation pane highlight the PowerPlex 21 panels folder that you just imported in Step 5 7 Select File then Import Bin Set 8 Navigate to the bins text file downloaded 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 G Creating a Size Standard with GeneMapper ID Software Version 3 2 There are two options when creating a size standard Use this protocol or the alternative protocol in Section 6 H 1 Select Tools then GeneMapper Manager 2 Select the Size Standard tab 3 Select New 4 Select Basic or Advanced Figure 17 The type of analysis method selected must match the type of analysis method created earlier Select OK Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD034 Printed in USA Revised 7 14 Page 38 5725TA Figure 17 The Select Dye and Analysis Method window 5 Enter a detailed name such as CC5 ILS 60 to 500 in the Siz
16. 4 3 16 27 Krenke B E et al 2005 Development of a novel fluorescent two primer approach to quantitative PCR Profiles in DNA 8 1 3 5 9 Appendix 9 A Advantages of Using the Loci in the PowerPlex 21 System The loci included in the PowerPlex 21 System Tables 4 and 5 were selected because they meet the needs of forensic laboratories that commonly genotype samples from Chinese populations The PowerPlex 21 System amplifies all loci commonly used in Chinese forensic genotyping laboratories in a single reaction Table 6 lists the PowerPlex 21 System alleles revealed in commonly available standard DNA templates We have carefully selected primers to avoid or minimize artifacts including those associated with DNA polymerases such as repeat slippage and terminal nucleotide addition 14 15 Repeat slippage sometimes called n 4 peaks stutter or shadow peaks 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 16 17 occurs when a thermostable nonproofeading 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 peak one base shorter than expected
17. 5 963Mb TCTA Complex 19 D21S11 TMR ET 21q21 1 19 476Mb TCTA Complex 19 D7S820 TMR ET 7q21 11 83 433Mb GATA D5S818 TMR ET 5q23 2 123 139Mb AGAT TPOX TMR ET 2p25 3 1 472Mb AATG D8S1179 CXR ET 8q24 13 125 976Mb TCTA Complex 19 D12S391 CXR ET 12p12 12 341Mb AGAT AGAC Complex D19S433 CXR ET 19q12 35 109Mb AAGG Complex FGA CXR ET 4q28 155 866Mb TTTC Complex 19 1Information about these chromosomal location of these loci can be found in references 20 21 and 22 and at www cstl nist gov biotech strbase chrom htm 2The 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 loci not associated with a coding gene the first database entry or original literature description shall be used 3Amelogenin is not an STR but displays an 89 base X specific band and a 95 base Y specific band NA Not applicable Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD034 Printed in USA Revised 7 14 Page 62 Table 5 The PowerPlex 21 System Allelic Ladder Information STR Locus Label Size Range of Allelic Ladder Comp
18. Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD034 Revised 7 14 Page 35 9776TA Figure 16 The GeneMapper ID X Allele tab 6 E Creating a Databasing or Paternity Analysis Method with GeneMapper ID X Software Version 1 2 continued 3130xl Genetic Analyzers For the Applied Biosystems 3500 and 3500xL Genetic Analyzers Life Technologies suggests an analysis threshold of 175RFU under their default injection conditions However individual laboratories should determine their peak amplitude thresholds from internal validation studies Peak heights for the CC5 ILS 500 are generally lower than those for the other dyes Therefore the threshold for the orange dye may be lower than that for the other dyes 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 10 and 11 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 Processing Data for Databasing or Paternity Samples 1 Select File then New Project 2 Select Edit then Add Sa
19. TMD034 Printed in USA Revised 7 14 Page 4 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD034 Revised 7 14 3 B Spectral Calibration Proper spectral calibration is critical to evaluate multicolor systems with the ABI PRISM 3100 and 3100 Avant Genetic Analyzers and Applied Biosystems 3130 3130xl 3500 and 3500xL Genetic Analyzers A matrix must be generated for each individual instrument For protocols and additional information on spectral calibration see the PowerPlex 5 Dye Matrix Standards 3100 3130 Technical Bulletin TBD024 This manual is available online at www promega com protocols 4 Protocols for DNA Amplification Using the PowerPlex 21 System The PowerPlex 21 System is optimized for the GeneAmp PCR System 9700 thermal cycler The use of gloves and aerosol resistant pipette tips is highly recommended to prevent cross contamination Keep all pre 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 The concentration of 2800M Control DNA was determined by measuring absorbance at 260nm
20. V Germany e Allele sequences for one or more of the loci vWA FGA D8S1179 D21S11 and D18S51 in allelic ladder mixtures is licensed under U S Pat Nos 7 087 380 7 645 580 Australia Pat No 2003200444 and corresponding patent claims outside the US f TMR ET CXR ET and CC5 dyes are proprietary 9 F Summary of Changes The following change was made to the 7 14 revision of this document Legal disclaimers were updated and discontinued products were removed Page 68 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD034 Printed in USA Revised 7 14 g This product or portions thereof is manufactured and sold under license from GE Healthcare under Australia Pat No 692230 Austria Pat No E236994 Belgium Pat No 0743987 Canada Pat No 2231475 EP Pat Nos 0743987 and 0851867 France Pat Nos 0743987 and 0851867 Germany Pat Nos 19581489 69530286 8 and 0851867 Italy Pat Nos 0743987 and 0851867 Japan Pat No 3066984 Liechtenstein Pat Nos 0743987 and 0851867 Netherlands Pat Nos 0743987 and 0851867 Spain Pat Nos 2197193 and 2173310 Sweden Pat Nos 0743987 and 0851867 Switzerland Pat Nos 0743987 and 0851867 United Kingdom Pat Nos 0743987 and 0851867 U S Pat Nos 5 654 419 5 688 648 5 869 255 6 177 247 5 707 804 6 028 190 6 544 744 7 015 000 and 5 728 528 and other pe
21. al 1998 New reference allelic ladders to improve allelic designation in a multiplex STR system Int J Legal Med 111 267 72 20 Butler J M 2006 Genetics and genomics of core STR loci used in human identity testing J Forensic Sci 51 253 65 21 Hill C R et al 2008 Characterization of 26 miniSTR loci for improved analysis of degraded DNA samples J Forensic Sci 53 73 80 22 Lu D J Liu Q L and Zhao H 2011 Genetic data of nine non CODIS STRs in Chinese Han population from Guangdong Province Southern China Int J Legal Med 125 133 7 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD034 Revised 7 14 8 References continued 23 B r W et al 1997 DNA recommendations Further report of the DNA Commission of the ISFH regarding the use of short tandem repeat systems Int J Legal Med 110 175 6 24 Gill P et al 1997 Considerations from the European DNA Profiling Group EDNAP concerning STR nomenclature Forensic Sci Int 87 185 92 25 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 26 Mandrekar P V Krenke B E and Tereba A 2001 DNA IQ The intelligent way to purify DNA Profiles in DNA
22. bring storage card punches to the bottom of the wells and remove any air bubbles Thermal Cycling Amplification and detection instrumentation may vary You will need to optimize protocols including cycle number 24 27 cycles and injection conditions for each laboratory instrument Testing at Promega shows that 25 cycles works well for a varity of sample types Buccal samples may require more amplification cycles than blood samples Cycle number will need to be optimized in each laboratory for each sample type that is amplified 1 Place the MicroAmp plate in the thermal cycler 2 Select and run the recommended protocol The preferred protocol for use with the GeneAmp PCR System 9700 thermal cycler is provided below The estimated total cycle time is 1 5 hours 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 Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD034 Printed in USA Revised 7 14 Page 10 Thermal Cycling Protocol1 96 C for 1 minute then 94 C for 10 seconds 59 C for 1 minute 72 C for 30 seconds for 25 cycles then 60 C for 20 minutes 4 C soak 1When using the GeneAmp PCR System 9700 thermal cycler the program mu
23. final reaction volume of 25 l Decreasing the reaction volume may result in suboptimal performance Improper storage of the 2800M Control DNA Thermal cycler plate or tube problems Review the thermal cycling protocol in Section 4 We have not tested other reaction tubes plates or thermal cyclers Calibrate the thermal cycler heating block if necessary Primer concentration was too low Use the recommended primer concentration Vortex the PowerPlex 21 5X Primer Pair Mix for 15 seconds before use Poor capillary electrophoresis injection CC5 ILS 500 peaks also affected Re inject the sample Check the syringe for leakage Check the laser power Samples were not denatured completely Heat denature samples for the recommended time then cool on crushed ice or in an ice water bath immediately prior to 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 Extra peaks visible in one Contamination with another template DNA or previously or all color channels amplified DNA Cross contamination can be a problem Use aerosol resistant pipette tips and change gloves regularly Samples were not denatured completely Heat denature samples for the recommended time and cool on crushed ice or in an ice water bath immediately prior to capillary electrophoresis Do not cool samples in a therma
24. 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 or imported in Section 6 C 8 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 9 If all analysis requirements are met the Save Project window will open Figure 15 Page 33 9430TA Figure 15 The Save Project window 6 D Creating a Casework Analysis Method with GeneMapper ID X Software Version 1 2 continued 10 Enter the project name 11 Choose the applicable security group from the drop down menu then select OK Note Sizing of alleles 475 bases will not use Local Southern Method For Penta E alleles gt 24 will be labeled as OL When the analysis is finished the Analysis Summary screen will appear We recommend that
25. less than 1 5 hours 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 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 four 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 24 27 cycles 4 Following amplification use your laboratory s validated separation and detection protocols to determine the optimal cycle number for the sample type Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD034 Revised 7 14 Page 13 Thermal Cycling Protocol1 96 C for 1 minute then 94 C for 10 seconds 59 C for 1 minute 72 C for 30 seconds for 25 cycles then 60 C for 20 minutes 4 C soak 1When using the GeneAmp PCR System 9700 thermal cycler the program must be run with Max mode as the ramp
26. one CC5 ILS 500 fragment smaller than the smallest sample peak or allelic ladder peak and at least one CC5 ILS 500 fragment larger than the largest sample peak or allelic ladder peak Run was too short and larger peaks in ILS were not captured Not all CC5 ILS 500 peaks defined in the size standard were detected during the run Create a new size standard using the internal lane standard fragments present in the sample Re run samples using a longer run time Off ladder alleles An allelic ladder from a different run than the samples was used Re analyze samples with an allelic ladder from the same run The GeneMapper ID software requires that the allelic ladder be imported from the same folder as the sample Be sure that the allelic ladder is in the same folder as the sample Create a new project and re analyze as described in Section 6 I or 6 J Panels text 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 Starting data point was incorrect for the partial range chosen correctly
27. post PCR cleanup or desalting lower conductivity formamide or reduced amounts of CC5 ILS 500 In house validation should be performed for any of these methods The PowerPlex 21 5X Master Mix was not vortexed well before use Vortex the 5X Master Mix for 5 10 seconds before dispensing into the PCR amplification mix Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD034 Printed in USA Revised 7 14 Page 48 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD034 Revised 7 14 Symptoms Causes and Comments Faint or absent allele peaks An air bubble formed at the bottom of the reaction tube Use a continued 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 The reaction volume was too low This system is optimized for a
28. promega com Printed in USA Part TMD034 Revised 7 14 Page 11 4 C Direct Amplification of DNA from Swabs continued 3 Use a clean MicroAmp plate for reaction assembly and label appropriately 4 Add the final volume of each reagent listed in Table 3 to a sterile tube 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 5 0ng l and add 2 l to a reaction well containing 23 l of PCR amplification 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 or PunchSolution Reagent is processed as a blank without a swab 9 Seal the plate Optional Briefly centrifuge the plate to bring contents t
29. seen in the n 2 and n 2 positions with some loci such as D1S1656 D6S1043 D13S317 vWA D21S11 D7S820 D5S818 D12S391 and D19S433 N 1 peaks are sometimes present at amelogenin Artifact peaks may be seen in the fluorescein channel at 66 69 bases in the JOE channel at 60 62 bases and 82 83 bases in the TMR channel at 60 67 bases and in the CXR channel at 58 65 bases and 76 77 bases During the course of species testing we noted that pig DNA yields a peak at 364 366 bases at the junction between CSF1PO and Penta D 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 This section provides information about general amplification and detection For questions about direct amplification see Sections 7 B and 7 C Symptoms 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 if available Insufficient template Low copy number LCN analysis using capillary electrophoresis may benefit from reducing competing charged particles during injection This can be accomplished with
30. 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 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 20 minute extension step at 60 C after thermal cycling Section 4 C Use 2 l of swab extract in a 25 l reaction A larger volume of swab extract may contain more than the recommended amount of DNA template resulting in incomplete adenylation Decrease cycle number Increase the final extension time 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 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 allele
31. 4 4330 Fax 608 277 2516 www promega com Part TMD034 Printed in USA Revised 7 14 Page 64 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD034 Revised 7 14 9 C The CC5 Internal Lane Standard 500 The CC5 Internal Lane Standard 500 contains 21 DNA fragments of 60 65 80 100 120 140 160 180 200 225 250 275 300 325 350 375 400 425 450 475 and 500 bases in length Figure 24 Each fragment is labeled with CC5 dye and can be detected separately as a fifth color in the presence of PowerPlex 21 amplified material The CC5 ILS 500 is designed for use in each CE injection to increase precision in analyses when using the PowerPlex 21 System Protocols to prepare and use this internal lane standard are provided in Section 5 Note Sizing of alleles 475 bases will not use Local Southern Method For Penta E alleles gt 24 will be labeled as OL 9 D Composition of Buffers and Solutions Page 65 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 base 0 037g EDTA Na2EDTA 2H2O 20 g ml glycogen Dissolve T
32. 5 Internal Lane Standard 500 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 and linker also affect 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 an 89 base X specific band and a 95 base Y specific band Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD034 Revised 7 14 Page 63 Table 6 The PowerPlex 21 System Allele Determinations in Commonly Available Standard DNA Templates STR Locus Standard DNA Templates1 2800M 9947A 9948 Amelogenin X Y X X X Y D3S1358 17 18 14 15 15 17 D1S1656 12 13 18 3 18 3 14 17 D6S1043 12 20 12 18 12 12 D13S317 9 11 11 11 11 11 Penta E 7 14 12 13 11 11 D16S539 9 13 11 12 11 11 D18S51 16 18 15 19 15 18 D2S1338 22 25 19 23 23 23 CSF1PO 12 12 10 12 10 11 Penta D 12 13 12 12 8 12 TH01 6 9 3 8 9 3 6 9 3 vWA 16 19 17 18 17 17 D21S11 29 31 2 30 30 29 30 D7S820 8 11 10 11 11 11 D5S81
33. 6 In the navigation pane highlight the PowerPlex 21 panels folder that you just imported in Step 5 7 Select File then Import Bin Set 8 Navigate to the bins text file downloaded in the Getting Started Section Select the file then Import 9 In the navigation pane highlight the PowerPlex 21 panels folder that you just imported in Step 5 10 Select File then Import Marker Stutter A warning box will appear asking if you want to overwrite current values Select Yes 11 Navigate to the stutter text file 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 6 B Creating a Size Standard with GeneMapper ID X Software Version 1 2 There are two options when creating a size standard Use this protocol or the alternative protocol in Section 6 C 1 Select Tools then GeneMapper ID X Manager 2 Select the Size Standard tab 3 Select New 4 In the Size Standard Editor window Figure 12 select GeneMapper ID X Security Group as the Security Group This allows access for all users of the software Other security groups may be used 5 Enter a detailed name such as CC5_ILS_500_IDX 6 Choose Orange for the Size Standard Dye Promega Corporation 2800 Woods Hollow Road
34. 8 12 12 11 11 11 13 TPOX 11 11 8 8 8 9 D8S1179 14 15 13 13 12 13 D12S391 18 23 18 20 18 24 D19S433 13 14 14 15 13 14 FGA 20 23 23 24 24 26 1Information on 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 Information about the use of 9947A and 9948 DNA as standard DNA templates can be found in reference 25 9 B 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 The SwabSolution Kit Cat DC8271 contains reagents for rapid DNA preparation from single source buccal swab samples prior to PowerPlex System analysis 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 DNA IQ System Cat DC6700 is a DNA isolation system designed specifically for forensic and paternity samples 26 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 frequ
35. 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 4 203 21 15 Schl tterer 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 Griffiths R et
36. Amplified Fragments Using the Applied Biosystems 3500 or 3500xL Genetic Analyzer continued 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 6 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 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD034 Printed in USA Revised 7 14 Page 20 Figure 6 The Create New Assay window 9229TA 6 To create a new File Name Convention Figure 7 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 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD034 Revised 7 14 Page 21 Figure 7 The Create New File Name Convention window 9252TA 5 A Detection of Amplified Fragments Using the Applied Biosystems 3500 or 3500
37. DNA Use the recommended amount of template DNA if available Stochastic effects can occur when amplifying low amounts of template The reaction volume was too low This system is optimized for a final reaction volume of 25 l to overcome inhibitors present in DNA samples Decreasing the reaction volume can result in suboptimal performance Miscellaneous balance problems Thaw the 5X Primer Pair Mix and 5X Master Mix completely and vortex for 15 seconds before use Note that the 5X Master Mix will take longer to thaw than the 5X Primer Pair Mix Do not centrifuge the 5X Primer Pair Mix or 5X Master Mix after mixing Calibrate thermal cyclers and pipettes routinely PCR amplification mix prepared in Section 4 was not mixed well Vortex the PCR amplification mix for 5 10 seconds before dispensing into the reaction tubes or plate Impure template DNA Inhibitors that may be present in forensic samples can lead to allele dropout or imbalance 7 B Direct Amplification of DNA From Storage Card Punches The following information is specific to direct amplification of DNA from storage card punches For additional information about general amplification and detection see Section 7 A Symptoms Causes and Comments Faint or absent allele peaks The reaction volume was too low This system is optimized for a final reaction volume of 25 l to overcome inhibitors present in FTA cards and PunchSolution Reagent Decreasing the reaction volume may resul
38. Ladder Mix is provided in a separate sealed bag for shipping This component should be moved to the post amplification box after opening The Water Amplification Grade is provided in a separate sealed bag for shipping This component should be moved to the pre amplification box after opening Amplification Setup Thermal Cycling Instrument Setup and Sample Preparation Data Analysis Section 4 Section 5 Section 6 Section 4 GeneAmp PCR System 9700 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 Figure 1 An overview of the PowerPlex 21 System protocol Applied Biosystems 3500 or 3500xL Genetic Analyzer Section 5 A GeneMapper ID Software Version 3 2 GeneMapper ID X Software Version 1 2 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD034 Revised 7 14 Page 3 2 Product Components and Storage Conditions continued Storage Conditions For long term storage store all components except the 2800M Control DNA at 30 C to 10 C in a nonfrost free freezer Store the 2800M Control DNA at 2 10 C For daily use the PowerPlex 21 System components can be stored for up to 1 wee
39. Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD034 Printed in USA Revised 7 14 Page 28 7 Enter the sizes of the internal lane standard fragments 60 65 80 100 120 140 160 180 200 225 250 275 300 325 350 375 400 425 450 475 and 500 bases See Section 9 C Figure 24 8 Select OK Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD034 Revised 7 14 Page 29 8257TA Figure 12 The GeneMapper ID X Size Standard Editor 6 C Importing the CC5 ILS 500 IDX Size Standard into GeneMapper ID X Software Version 1 2 The CC5_ILS_500_IDX xml file is available for download at www promega com resources tools genemapper id software panels and bin sets Save the CC5_ILS_500_IDX xml file to a known location on your computer 1 Select Tools then GeneMapper ID X Manager 2 Select the Size Standard tab 3 Select Import 4 Navigate to the location of the CC5_ILS_500_IDX xml file on your computer 5 Highlight the file then select Import 6 Select Done to save changes and close the GeneMapper ID X Manager 6 D Creating a Casework Analysis Method with GeneMapper ID X Software Version 1 2 These instructions are intended as a guide
40. Revised 7 14 TMD034 PowerPlex 21 System Instruc ons for use of Products DC8902 AND DC8942 T E C H N I C A L M A N U A L PowerPlex 21 System All technical literature is available on the Internet at http www promega com protocols Please visit the web site to verify that you are using the most current version of this Technical Manual Please contact Promega Technical Services if you have questions on use of this system E mail genetic promega com Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD034 Revised 7 14 1 Description 2 2 Product Components and Storage Conditions 3 3 Before You Begin 4 A Precautions 4 B Matrix Standardization or Spectral Calibration 5 4 Protocols for DNA Amplification Using the PowerPlex 21 System 5 A Amplification of Extracted DNA
41. ak heights the deposition of cells onto a punch resulting in peak height variability between samples The PunchSolution Kit maximizes the recovery of amplifiable DNA from samples but does not normalize the amount of DNA present 7 C Direct Amplification of DNA From Swabs The following information is specific to direct amplification of DNA from swabs after pretreatment using the SwabSolution Kit For information about general amplification and detection see Section 7 A Symptoms Causes and Comments Faint or absent allele peaks Poor sample deposition Shedding and collection of donor cells was variable Increase cycle number Inactive SwabSolution Reagent Thaw the SwabSolution Reagent completely in a 37 C water bath and mix by gentle inversion Store the SwabSolution Reagent at 2 10 C Do not store reagents in the refrigerator door where the temperature can fluctuate Do not refreeze avoid multiple freeze thaw cycles as this may reduce activity Active 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 reagent 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 Use only a heat block to maintain effi
42. 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 D or 6 E 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 Starting data point was incorrect for the partial range chosen correctly in Section 6 E Adjust the starting data point in the analysis method Alternatively use a full range for the analysis Extra peaks in size standard Open the Size Match Editor Highlight the extra peak select Edit and select delete size label Select auto adjust sizes Run was too short and larger peaks in ILS were not cap
43. alyzed as described in the Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 1 Open the 3500 Data Collection Software The Dashboard screen will launch Figure 2 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 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 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD034 Printed in USA Revised 7 14 Page 16 9247TA Figure 2 The Dashboard Figure 3 shows the settings used at Promega for the Applied Biosystems 3500xL Genetic Analyzer for the application type dye set capillary length polymer run module and appropriate protocol information The only setting that was changed from the default settings is dye set The recommended settings are When creating an Instrument Protocol be sure to select the same dye set that was used to perform the Promega 5 dye spectral calibration We recommend using a run time of 1 210 1 500 seconds and the default injection conditions Run time and other instrument settings should be optimized a
44. alyzed 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 21 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 ET labeled allelic ladder components and their allele designations Panel D The CXR ET labeled allelic ladder components and their allele designations 6 L Results continued 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 The mean plus three standard deviations at each locus is used in the PowerPlex 21 panels text file for locus specific filtering in the GeneMapper ID software version 3 2 and in the PowerPlex 21 stutter text file for locus specific filtering in GeneMapper ID X software In addition to stutter peaks other artifact peaks can be observed at some of the PowerPlex 21 System loci Low level products can be
45. ation 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
46. bubbles Thermal Cycling 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 30 cycles work well for 0 5ng of purified DNA templates 1 Place the MicroAmp plate or reaction tubes in the thermal cycler 2 Select and run the recommended protocol The preferred protocol for use with the GeneAmp PCR System 9700 thermal cycler is provided below The estimated total cycling time is 1 5 hours 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 Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD034 Revised 7 14 Page 7 Thermal Cycling Protocol1 96 C for 1 minute then 94 C for 10 seconds 59 C for 1 minute 72 C for 30 seconds for 30 cycles then 60 C for 10 minutes 4 C soak 1When using the GeneAmp PCR System 9700 thermal cycler the program must be run with Max mode as the ramp speed This requires a silver or gold plated silver sample block The ramp speed is set after the thermal cycling run is started The Select Method Options screen appears Select
47. can indicate the presence of double stranded DNA due to incomplete denaturation or post injection re annealing CE related artifacts spikes Minor voltage changes or urea crystals passing by the laser can cause spikes or unexpected peaks Spikes sometimes appear in one color but often are easily identified by their presence in more than one color Re inject samples to confirm Incorrect G5 spectral was active Re run samples and confirm that the PowerPlex 5 dye G5 spectral is set for G5 See instructions on instrument preparation in Section 5 Pull up or bleedthrough Pull up can occur when peak heights are too high or if a poor or incorrect matrix is applied to the samples Perform a new spectral calibration and re run the samples Instrument sensitivities can vary Optimize the injection conditions See Section 5 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 fluorescein and JOE channels Use autoclaved water change vials and wash buffer reservoir Repeat sample preparation using fresh formamide Long term storage of amplified sample in formamide can result in artifacts The CE polymer was beyond its expiration date or polymer was stored at room temperature for more than one week Maintain instrumentation on a daily or weekly basis as recommended by the manufacturer Allelic ladder not running Allelic lad
48. cient heat transfer We have tested 60 minute incubation times and observed no difference in performance compared to a 30 minute incubation Faint or absent peaks for the If the positive control reaction failed to amplify check to positive control reaction 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 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 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD034 Revised 7 14 Page 53 7 C Direct Amplification of DNA From Swabs continued Symptoms Causes and Comments Extra peaks visible in one or Swab extract was contaminated Include a blank swab as a all color channels negative control when processing samples Artifacts of STR amplification Amplification of swab extracts with high DNA concentrations can result in artifact peaks due to overamplification resulting in saturated signal on the CE instrument We recommend 2 l of
49. 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 21 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_21_Panels_IDX_vX x txt PowerPlex_21_Bins_IDX_vX x txt and PowerPlex_21_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 Page 27 6 A PowerPlex 21 Panels Bins and Stutter Text Files with GeneMapper ID X Software Version 1 2 continued Importing Panels Bins and Stutter Text Files 1 Open the GeneMapper ID X software 2 Select Tools then Panel Manager 3 Highlight the Panel Manager icon in the upper left navigation pane 4 Select File then Import Panels 5 Navigate to the panels text file downloaded in the Getting Started Section Select the file then Import
50. d procedures for amplification and fluorescence detection Additional research and validation are required if any modifications to the recommended protocols are made 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 21 5X Master Mix PowerPlex 21 5X Primer Pair Mix 2800M Control DNA and Water Amplification Grade are provided in a separate box and should be stored separately from those used following amplification PowerPlex 21 Allelic Ladder Mix and CC5 Internal Lane Standard 500 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 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part
51. der and primer pair mix were not compatible Ensure the same as samples that the allelic ladder is from the same kit as the primer pair mix Poor quality formamide Use only Hi Di formamide when analyzing samples Be sure the allelic ladder and samples are from the same instrument run Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD034 Printed in USA Revised 7 14 Page 50 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD034 Revised 7 14 Symptoms Causes and Comments Allelic ladder not running Migration of samples changed slightly over the course of a the same as samples continued CE run with many samples This may be due to changes in temperature or the CE column over time Use a different injection of allelic ladder to 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 0 5ng of template can result in an imbalance with smaller loci showing more product than larger loci Decrease number of cycles Degraded DNA sample DNA template was degraded and larger loci showed diminished yield Repurify template DNA if possible Insufficient template
52. 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 F 7 In the Size Standard column select the size standard that was created in Section 6 G or imported in Section 6 H 8 Select Analyze green arrow button to start the data analysis Note Sizing of alleles 475 bases will not use Local Southern Method For Penta E alleles gt 24 will be labeled as OL 6 K 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 DNA allelic repeat sizes with the locus specific allelic ladder The expected 2800M DNA allele designations for each locus are listed in Table 6 Section 9 A 6 L Results Representative results of the PowerPlex 21 System are shown in Figure 22 The PowerPlex 21 Allelic Ladder Mix is shown in Figure 23 Page 45 Page 46 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA
53. e Cat DNA IQ System 100 reactions DC6701 400 reactions DC6700 Plexor HY System 200 reactions DC1001 800 reactions DC1000 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD034 Printed in USA Revised 7 14 Page 66 Page 67 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD034 Revised 7 14 a U S Pat No 6 242 235 Australian Pat No 761757 Canadian Pat No 2 335 153 Chinese Pat No ZL99808861 7 Hong Kong Pat No HK 1040262 Japanese Pat No 3673175 European Pat No 1088060 and other patents pending b U S Pat Nos 5 843 660 6 479 235 6 221 598 and 7 008 771 Australian Pat No 724531 Canadian Pat No 2 118 048 and 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 U S Pat No 6 238 863 European Pat No 1058727 Chinese Pat No ZL99802696 4 Japanese Pat No 4494630 and other patents pending d STR loci are the subject of U S Pat No RE 37 984 German Pat No DE 38 34 636 C2 and other patents issued to the Max Planck Gesellschaft zur F rderung der Wissenschaften e
54. e injection time injection voltage or the amount of sample 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 to achieve the desired signal intensity If the injection time or voltage is reduced a decreased peak amplitude threshold for the orange channel may be required for proper sizing 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 Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD034 Revised 7 14 Page 15 5 A Detection of Amplified Fragments Using the Applied Biosystems 3500 or 3500xL Genetic Analyzer continued 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 an
55. e Standard Editor Figure 18 6 Choose Orange for the Size Standard Dye 7 Enter the sizes of the internal lane standard fragments 60 65 80 100 120 140 160 180 200 225 250 275 300 325 350 375 400 425 450 475 and 500 bases See Section 9 C Figure 24 8 Select OK Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD034 Revised 7 14 Page 39 8199TA Figure 18 The Size Standard Editor 6 H Importing the CC5 ILS 500 Size Standard into GeneMapper ID Software Version 3 2 The CC5_ILS_500 xml file is available for download at www promega com resources tools genemapper id software panels and bin sets Save the CC5_ILS_500 xml file to a known location on your computer 1 Select Tools then GeneMapper Manager 2 Select the Size Standard tab 3 Select Import 4 Browse to the location of the CC5_ILS_500 xml file 5 Highlight the file then select Import 6 Select Done to save changes and exit the GeneMapper Manager 6 I 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
56. e of Classic mode analysis on samples can result in baselines with more noise than those analyzed using the Basic or Advanced mode analysis method Advanced mode analysis methods and size standards are recommended Incorrect G5 spectral was active Re run samples and confirm that the PowerPlex 5 dye G5 spectral is set for G5 See instructions for instrument preparation in Section 5 Error message after attempting There was a conflict between different sets of panels and bins to import panels and bins text files text files Check to be sure that the bins are installed properly Unable to save panel data If not delete all panels and bins text files and re import files java SQLEException in a different order ORA 00001 unique constraint IFA CKP_NNN violated Allelic ladder peaks GeneMapper ID software was not used or microsatellite labeled off ladder analysis settings were used instead of HID analysis settings GeneMapper software does not use the same algorithms as GeneMapper ID software and cannot correct for sizing differences using the allelic ladder Promega recommends using GeneMapper ID software to analyze PowerPlex reactions If using GeneMapper ID software version 3 2 be sure that the analysis method selected is an HID method This can be verified by opening the analysis method using the GeneMapper Manager then selecting the General tab The analysis type cannot be changed If the method is not HID it sh
57. e standard mode Classic vs Basic or Advanced Be sure both files or analysis method is invalid are set to the same mode either Classic or Basic or Advanced mode No alleles called but no error Panels text file was not selected for sample In the Panel message appears 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 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 The bins text file assigned to the analysis method was deleted Both the Bin Set used in the In the GeneMapper Manager select the Analysis Methods tab Analysis Method and the Panel and open the analysis method of interest Select the Allele tab must belong to the same and select an appropriate bins text file Chemistry Kit 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 19 Significantly raised baseline Poor spectral calibration Perform a new spectral calibration and re run the samples Use of Classic mode analysis method Us
58. e the GeneMapper ID software Contact Applied Biosystems 5 Enter a descriptive name for the analysis method such as PowerPlex21_20 filter 6 Select the Allele tab Figure 21 7 Select the bins text file that was imported in Section 6 F 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 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD034 Revised 7 14 Page 43 6 J Creating a Databasing or Paternity Analysis Method with GeneMapper ID Software Version 3 2 continued 9 Enter the values shown in Figure 21 for proper filtering of peaks when using the PowerPlex 21 System For an explanation of the proper usage and effect of these settings refer to the Applied Biosystems user bulletin titled Installation Procedures and New Features for GeneMapper ID Software 3 2 10 Select the Peak Detector tab We recommend the settings shown in Figure 20 Notes 1 Select full range or partial range for the analysis range When using a partial range choose an appropriate analysis range based on your data Choose a start point after the 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 tha
59. e the final extension time Peak height imbalance Excessive amount of DNA Amplification of gt 20ng of template can result in an imbalance 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 card Decrease cycle number Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD034 Printed in USA Revised 7 14 Page 52 Symptoms Causes and Comments Peak height imbalance continued The reaction volume was too low This system is optimized for a final reaction volume of 25 l to overcome inhibitors present in FTA cards and PunchSolution Reagent Decreasing the reaction volume can result in suboptimal performance Amplification was inhibited when using more than one storage card punch with blood Use only one 1 2mm storage card punch with blood Bode Buccal DNA Collector devices were used without a lysis step For buccal samples on Bode Buccal DNA Collector devices we recommend pretreatment with the PunchSolution Reagent to lyse samples before adding the amplification mix Extreme variability in sample There can be significant individual to individual variability in to sample pe
60. e use of the products including without limitation any claim of inaccurate invalid or incomplete results Exclusion of Liability GE Healthcare Bio Sciences Corp and its affiliates shall have no liability to an End User including without limitation for any loss of use or profits business interruption or any consequential incidental special or other indirect damages of any kind regardless of how caused and regardless of whether an action in contract tort strict product liability or otherwise 2011 2012 2014 Promega Corporation All Rights Reserved Plexor and PowerPlex are registered trademarks of Promega Corporation DNA IQ Identity Automation PunchSolution and SwabSolution are trademarks of Promega Corporation ABI PRISM GeneMapper and MicroAmp are registered trademarks of Applera Corporation ART is a registered trademark of Molecular Bio Products Inc Bode Buccal DNA Collector is a trademark of the Bode Technology Group Inc EasiCollect and OmniSwab are trademarks of Whatman FTA is a registered trademark of Flinders Technologies Pty Ltd and is licensed to Whatman GeneAmp is a registered trademark of Roche Molecular Systems Inc Hi Di POP 4 and POP 7 are trademarks of Applera Corporation Sampact is a trademark of Fitzco Vacutainer is a registered trademark of Becton Dickinson and Company Products may be covered by pending or issued patents or may have certain limitations Please visit our Web site for more inform
61. ently encountered in casework samples With DNA rich samples the DNA IQ System delivers a consistent amount of total DNA The system has been used to isolate 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 Additional ordering information is available in Section 9 E For applications requiring human specific DNA quantification the Plexor HY System Cat DC1000 was developed 27 This qPCR based method provides total human and male specific DNA quantification in one reaction Additionally the Plexor HY System provides a post amplification melt analysis to confirm positive results and and Internal PCR Control IPC to confirm negative results Additional ordering information is available in Section 9 E 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 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 27
62. 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 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 F 7 In the Size Standard column select the size standard that was created in Section 6 G or imported in Section 6 H 8 Select Analyze green arrow button to start data analysis Note Sizing of alleles 475 bases will not use Local Southern Method For Penta E alleles gt 24 will be labeled as OL 6 J 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 hav
63. ftware Version 2 0 or the Applied Biosystems 3130 or 3130xl Genetic Analyzer with Data Collection Software Version 3 0 We do not recommend use of this product with the POP 7 polymer due to artifacts that may migrate within the fluorescein and JOE channels 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 polymer for the 3100 or 3130 10X genetic analyzer buffer with EDTA MicroAmp optical 96 well plate and septa or equivalent Hi Di formamide Applied Biosystems Cat 4311320 PowerPlex 5 Dye Matrix Standards 3100 3130 Cat DG4700 The quality of formamide is critical Use Hi Di formamide Freeze formamide in aliquots at 20 C Multiple freeze thaw cycles or long term storage at 4 C may cause breakdown of formamide Poor quality formamide may contain ions that compete with DNA during injection which results in lower peak heights and reduced sensitivity A longer injection time may not increase the signal Formamide is an irritant and a teratogen avoid inhalation and contact with skin Read the warning label and take appropriate precautions when handling this substance Always wear gloves and safety glasses when working with formamide Sample Preparat
64. h use Do not centrifuge the 5X 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 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 Note For nonFTA card punches add the PCR amplification mix to the pretreated punches For FTA card punches add the storage card punch in Step 6 It also is acceptable to add the FTA card punch first then add the PCR amplification mix 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 into the appropriate wells of the reaction plate Promega Corporation 2800 Woods Hollow Road Madison
65. hort 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 21 System a g is used for human identification applications including forensic analysis relationship testing and research use The system allows co amplification and four color fluorescent detection of 21 loci 20 STR loci and Amelogenin including D1S1656 D2S1338 D3S1358 D5S818 D6S1043 D7S820 D8S1179 D12S391 D13S317 D16S539 D18S51 D19S433 D21S11 Amelogenin CSF1PO FGA Penta D Penta E TH01 TPOX and vWA The PowerPlex 21 System is compatible with the ABI PRISM 3100 and 3100 Avant Genetic Analyzers and Applied Biosystems 3130 3130xl 3500 and 3500xL Genetic Analyzers Amplification and detection instrumentation may vary You may need to optimize protocols including cycle number injection conditions and loading volume for each laboratory instrument In house validation should be performed The PowerPlex 21 System provides all materials necessary to amplify STR regions of human genomic DNA including a hot start thermostable DNA pol
66. i e missing the terminal addition is sometimes seen We have modified primer sequences and added a final extension step at 60 C 18 to the amplification protocols to provide conditions for essentially complete terminal nucleotide addition when recommended amounts of template DNA are used Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD034 Printed in USA Revised 7 14 Page 60 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD034 Revised 7 14 Page 61 Table 4 The PowerPlex 21 System Locus Specific Information STR Locus Label Chromosomal Location1 Repeat Sequence2 5 3 Amelogenin3 Fluorescein Xp22 1 22 3 and Y NA D3S1358 Fluorescein 3p21 31 45 557Mb TCTA Complex D1S1656 Fluorescein 1q42 228 972Mb TAGA Complex D6S1043 Fluorescein 6q15 92 449Mb AGAT D13S317 Fluorescein 13q31 1 81 62Mb TATC Penta E Fluorescein 15q26 2 95 175Mb AAAGA D16S539 JOE 16q24 1 84 944Mb GATA D18S51 JOE 18q21 33 59 1Mb AGAA 19 D2S1338 JOE 2q35 218 705Mb TGCC TTCC CSF1PO JOE 5q33 1 149 436Mb AGAT Penta D JOE 21q22 3 43 88Mb AAAGA TH01 TMR ET 11p15 5 2 149Mb AATG 19 vWA TMR ET 12p13 31
67. in Section 6 I 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 CC5 ILS 500 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 orange channel to include peaks If peaks are low quality redefine the size standard for the sample to skip these peaks Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD034 Revised 7 14 Page 57 Page 58 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD034 Printed in USA Revised 7 14 7 E GeneMapper ID Software continued Symptoms Causes and Comments Error message The size standard and analysis method were not in the same Either panel siz
68. ing a Casework Analysis Method with GeneMapper ID Software Version 3 2 continued 10 Select the Peak Detector tab We recommend the settings shown in Figure 20 Notes 1 Select full range or partial range for the analysis range When using a partial range choose an appropriate analysis range based on your data Choose a start point after the 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 Peak heights for the CC5 ILS 500 are generally lower than those for the other dyes Therefore the threshold for the orange dye may be lower than that for the other dyes 11 Select the Peak Quality tab You may change the settings for peak quality Note For Steps 11 and 12 see the GeneMapper ID user s manual for more information 12 Select the Quality Flags tab You may change these settings 13 Select OK to save your settings Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD034 Printed in USA Revised 7 14 Page 42 8187TA Figure 20 The GeneMapper ID Peak Detector tab Processing Data
69. ion 1 Prepare a loading cocktail by combining and mixing CC5 Internal Lane Standard 500 and Hi Di formamide as follows 2 0 l CC5 ILS 500 injections 10 0 l Hi Di formamide injections Note The volume of internal lane standard used in the loading cocktail can be decreased to adjust the intensity of the size standard peaks based on laboratory preferences Keep the volume of formamide at 10 0 l per well and adjust the volume added to the wells in Step 3 accordingly 2 Vortex for 10 15 seconds to mix 3 Pipet 12 l of formamide internal lane standard mix into each well 4 Add 1 l of amplified sample or 1 l of PowerPlex 21 Allelic Ladder Mix Cover wells with appropriate septa Note Instrument detection limits vary therefore injection time injection voltage or the amount of sample mixed with loading cocktail may need to be adjusted Use the Module Manager in the data collection software to modify the injection time or voltage in the run module see Instrument Preparation below If the injection time or voltage is reduced a decreased peak amplitude threshold for the orange channel may be required for proper sizing Page 25 5 B Detection of Amplified Fragments Using the ABI PRISM 3100 or 3100 Avant Genetic Analyzer with Data Collection Software Version 2 0 and the Applied Biosystems 3130 or 3130xl Genetic Analyzer with Data Collection Software Version 3 0 continued
70. ion plate Applied Biosystems aerosol resistant pipette tips SwabSolution Kit Cat DC8271 This section contains a protocol for amplifying DNA from swab extracts using the PowerPlex 21 System and GeneAmp PCR System 9700 thermal cycler Pretreat cotton swabs or OmniSwabs GE Healthcare with the SwabSolution Kit Cat DC8271 as described in the SwabSolution Kit Technical Manual TMD037 to generate a swab extract Be sure to include a blank swab as a negative control when processing samples Amplification Setup 1 Thaw the PowerPlex 21 5X Master Mix and PowerPlex 21 5X 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 5X 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 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www
71. it table Marker header bar for some loci When an edit is made to a locus the quality flags and marker are gray 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 An insufficient number of CC5 ILS 500 fragments was defined Be sure to define at least one CC5 ILS 500 fragment smaller than the smallest sample peak or allelic ladder peak and at least one CC5 ILS 500 fragment 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 CC5 ILS 500 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 A low quality allelic ladder was used during analysis Ensure that only high quality allelic ladders are used for analysis Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD034 Revised 7 14 Page 55 7 D GeneMapper ID X Software continued Symptoms Causes and Comments Off ladder alleles An
72. k at 2 10 C The PowerPlex 21 5X Primer Pair Mix PowerPlex 21 Allelic Ladder Mix and CC5 Internal Lane Standard 500 CC5 ILS 500 are light sensitive and must be stored in the dark We strongly recommend that pre amplification and post amplification reagents be stored and used separately with different pipettes tube racks etc Available Separately The proper panels and bins text files for use with GeneMapper ID and ID X software are available for download 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 provided separately and are available for ABI PRISM 3100 and 3100 Avant Genetic Analyzers and Applied Biosystems 3130 3130xl 3500 and 3500xL Genetic Analyzers PowerPlex 5 Dye Matrix Standards 3100 3130 Cat DG4700 3 Before You Begin 3 A Precautions The application of PCR based typing for forensic or paternity casework requires validation studies and quality control measures that are not contained in this manual 10 11 Guidelines for the validation process are published in the Internal Validation of STR Systems Reference Manual 12 The quality of purified DNA or direct amplification samples 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 recommende
73. l 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 peaks one base smaller than the allele due to incomplete addition of the 3 A residue Be sure to perform the 20 minute extension step at 60 C after thermal cycling Section 4 Decrease the number of cycles Plasticware can alter heat transfer during amplification and prevent full adenylation Increase the final extension time Page 49 7 A Amplification and Fragment Detection continued Symptoms Causes and Comments Extra peaks visible in one Artifacts of STR amplification Amplification of excess or all color channels continued amounts of purified DNA can result in a higher number of artifact peaks Use the recommended amount of template DNA See Section 6 L for additional information on stutter and artifacts Artifacts The signal strength of certain artifacts increases with storage of the amplification plate at 4 C see Table 5 sometimes in as short a time period as overnight but more commonly when left at 4 C for a few days We recommend storing amplification products at 20 C Double stranded DNA migrates faster than single stranded DNA during capillary electrophoresis Appearance of shadow peaks migrating in front of the main peaks especially if the shadow peaks are separated by the same distance as the main peaks in a heterozygote
74. ltiple freeze thaw cycles as this may reduce activity Faint or absent peaks for the If the positive control reaction failed to amplify check to positive control reaction make sure that the correct amount of 2800M Control DNA was added to the reaction We recommend 10ng of 2800M Control DNA per 25 l amplification reaction Do not include a blank punch in the positive control reaction Presence of a blank punch may inhibit amplification of 2800M Control DNA Optimize the amount of 2800M Control DNA for your thermal cycling conditions and laboratory preferences When using a reduced cycle number add 5 10ng of 2800M DNA to the positive control reaction Improper storage of the 2800M Control DNA Extra peaks visible in one Punch may be contaminated Take punches from blank paper or all color channels between samples Include a reaction with one or two blank punches as a negative control 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 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 the 20 minute extension step at 60 C after thermal cycling Section 4 B Decrease cycle number Increas
75. mp S 903 one punch per 25 l amplification reaction Pretreat nonFTA sample types with the PunchSolution Kit 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 Automated punchers also can be used to create sample disks Refer to the user s guide for your instrument for assistance with generating 1 2mm disks technical advice and troubleshooting information Note Static may be problematic when adding a punch to a well For FTA card punches adding PCR amplification mix to the well before adding the punch may help alleviate static problems Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD034 Printed in USA Revised 7 14 Page 8 Amplification Setup 1 Thaw the PowerPlex 21 5X Master Mix and PowerPlex 21 5X Primer Pair Mix completely Note Centrifuge tubes briefly to bring contents to the bottom then vortex reagents for 15 seconds before eac
76. mples to Project 3 Browse to the location of 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 In the Analysis Method column select the analysis method created above 5 In the Panel column select the panels text file that was imported in Section 6 A 6 In the Size Standard column select the size standard that was created in Section 6 B or imported in Section 6 C 7 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 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD034 Printed in USA Revised 7 14 Page 36 Promega Corporation 2800 Woods Holl
77. n mix 3 Use a clean MicroAmp plate for reaction assembly and label appropriately Alternatively determine the number of clean 0 2ml reaction tubes required and label appropriately 4 Add the final volume of each reagent listed in Table 1 into a sterile tube 5 Vortex the PCR amplification mix for 5 10 seconds then pipet the 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 Add template DNA 0 5ng for each sample to the respective well containing PCR amplification mix Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD034 Printed in USA Revised 7 14 Page 6 Table 1 PCR Amplification Mix for Amplification of Extracted DNA PCR Amplification Mix Component1 Volume Per Reaction Number of Reactions Final Volume Water Amplification Grade to a final volume of 25 0 l PowerPlex 21 5X Master Mix 5 0 l PowerPlex 21 5X Primer Pair Mix 5 0 l template DNA 0 5ng 2 3 up to 15 0 l total reaction volume 25 l 1Add Water Amplification Grade to the tube first then add PowerPlex 21 5X Master Mix and PowerPlex 21 5X Primer Pair Mix The template DNA will be added at Step 6 2Store DNA templates in nuclease free water
78. nd validated in your laboratory Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD034 Revised 7 14 Page 17 Figure 3 The Create New Instrument Protocol window 9393TA Application Type HID Capillary Length 36cm Polymer POP 4 Dye Set G5 Promega G5 spectral Run Module HID36_POP4 xl Injection Time1 24 seconds Injection Voltage 1 2kV Run Time 1 210 1 500 seconds 1Injection time may be modified 2 24 seconds to increase or decrease peak heights Page 18 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD034 Printed in USA Revised 7 14 5 A Detection of Amplified Fragments Using the Applied Biosystems 3500 or 3500xL Genetic Analyzer continued When optimizing injection conditions in your laboratory you may choose to create specific Instrument Protocols for each condition tested If a single Instrument Protocol is used follow the instructions in the Applied Biosystems 3500 3500xL Genetic Analyzers User Guide to edit a library entry Assign a descriptive protocol name Note For more detailed information refer to the Applied Biosystems 3500 3500xL Genetic Analyzers User Guide 3 To create a new Size Standa
79. nding and foreign patent applications End User Terms and Conditions Acceptance These terms and conditions shall govern the purchase use transfer and acceptance of the products described in the purchase order quotation or invoice which products are sold and distributed by Promega to the buyer transferee of such products the End User The transfer sale of products to the End User is expressly conditional upon End User s acceptance of these terms and conditions Restrictions on Use End Users are specifically not authorized to and are forbidden from reselling transferring or distributing any products either as a stand alone product or as a component of another product The right to use the products does not in and of itself include or carry any right of the End User to any GE Healthcare Bio Sciences Corp s technology or intellectual property other than expressly provided herein End Users may not use sequence s in an attempt to reverse engineer parameters of any of GE Healthcare Bio Sciences Corp proprietary products or services Disclaimer of Warranties GE Healthcare Bio Sciences Corp provides no warranties to end user statutory or implied including without limitation as to product quality condition description merchantability or fitness for a particular purpose and all such warranties are hereby expressly disclaimed GE Healthcare Bio Sciences Corp hereby expressly disclaims any warranty regarding results obtained through th
80. o the bottom of the wells and remove any air bubbles Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD034 Printed in USA Revised 7 14 Page 12 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 13 l PowerPlex 21 5X Master Mix 5 0 l PowerPlex 21 5X Primer Pair Mix 5 0 l swab extract 2 0 l total reaction volume 25 l 1Add Water Amplification Grade to the tube first then add PowerPlex 21 5X Master Mix and PowerPlex 21 5X Primer Pair Mix The swab extract will be added at Step 6 Thermal Cycling Amplification and detection instrumentation may vary You will need to optimize protocols including cycle number 24 27 cycles and injection conditions or loading volume for each laboratory instrument Testing at Promega shows that 25 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 1 Place the MicroAmp plate in the thermal cycler 2 Select and run the recommended protocol The preferred protocol for use with the GeneAmp PCR System 9700 thermal cycler is provided below The estimated total cycle time is
81. onents1 2 bases Repeat Numbers of Allelic Ladder Components3 Amelogenin4 Fluorescein 89 95 X Y D3S1358 Fluorescein 103 147 9 20 D1S1656 Fluorescein 161 208 9 14 14 3 15 15 3 16 16 3 17 17 3 18 18 3 19 19 3 20 3 D6S1043 Fluorescein 215 287 7 25 D13S317 Fluorescein 302 350 5 17 Penta E Fluorescein 371 466 5 24 D16S539 JOE 84 132 4 16 D18S51 JOE 134 214 7 10 10 2 11 13 13 2 14 27 D2S1338 JOE 224 296 10 12 14 28 CSF1PO JOE 318 362 5 16 Penta D JOE 377 450 2 2 3 2 5 17 TH01 TMR ET 72 115 3 9 9 3 10 11 13 3 vWA TMR ET 127 183 10 24 D21S11 TMR ET 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 D7S820 TMR ET 269 313 5 16 D5S818 TMR ET 321 369 6 18 TPOX TMR ET 393 441 4 16 D8S1179 CXR ET 76 124 7 19 D12S391 CXR ET 133 185 14 17 17 3 18 18 3 19 27 D19S433 CXR ET 193 245 5 2 6 2 8 12 12 2 13 13 2 14 14 2 15 15 2 16 16 2 17 17 2 18 18 2 FGA CXR ET 265 411 14 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 32 2 33 2 42 2 43 2 44 2 45 2 46 2 48 2 50 2 1The length of each allele in the allelic ladder has been confirmed by sequence analyses 2When using an internal lane standard such as the CC
82. ould be deleted and a new analysis method created Page 59 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 b actin related pseudogene 2 actbp2 detected using the polymerase chain reaction Nucleic Acids Res 19 6980 5 Ausubel F M et al 1996 Unit 15 The polymerase chain reaction In Current Protocols in Molecular Biology Vol 2 John Wiley and Sons NY 6 Sambrook J Fritsch E F and Maniatis T 1989 Chapter 14 In vitro amplification of DNA by 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
83. ow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD034 Revised 7 14 8 If all analysis requirements are met the Save Project window will open Figure 15 9 Enter the project name 10 Choose the applicable security group from the drop down menu then select OK Note Sizing of alleles 475 bases will not use Local Southern Method For Penta E alleles gt 24 will be labeled as OL 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 F PowerPlex 21 Panels and Bins Text Files with GeneMapper ID Software Version 3 2 To facilitate analysis of data generated with the PowerPlex 21 System we have created panels and bins text files to allow automatic assignment of geno
84. rd 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 PPlex_ILS500 or another appropriate name Choose Orange as the Dye Color The fragments in the size standard are 60 65 80 100 120 140 160 180 200 225 250 275 300 325 350 375 400 425 450 475 and 500 bases See Figure 4 Figure 4 The Create New Size Standard window 9227TA Page 19 4 To create a new QC Protocol navigate to the Library Select QC Protocols then select Create Alternatively 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 21 System on the Applied Biosystems 3500 or 3500xL Genetic Analyzer Figure 5 shows one option for these settings Note Peak heights for the CC5 ILS 500 are generally lower than those for the other dyes Therefore the threshold for the orange dye may be lower than that for the other dyes Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD034 Revised 7 14 Figure 5 The Create New QC Protocol window 9228TA 5 A Detection of
85. ris 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 Figure 24 CC5 Internal Lane Standard 500 An electropherogram showing the CC5 Internal Lane Standard 500 fragments 8248TA 9 E Related Products STR Systems Product Size Cat 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 16 HS System 100 reactions DC2101 400 reactions DC2100 PowerPlex 18D System 200 reactions DC1802 800 reactions DC1808 PowerPlex CS7 System 100 reactions DC6613 PowerPlex Y23 System 50 reactions DC2305 200 reactions DC2320 Not for Medical Diagnostic Use Accessory Components Product Size Cat PowerPlex 5 Dye Matrix Standards 3100 3130 25 l each dye DG4700 2800M Control DNA 10ng l 25 l DD7101 2800M Control DNA 0 25ng l 500 l DD7251 SwabSolution Kit 100 preparations DC8271 PunchSolution Kit 100 preparations DC9271 CC5 Internal Lane Standard 500 300 l DG1521 Water Amplification Grade 6 250 l 5 1 250 l DW0991 Not for Medical Diagnostic Use Sample Preparation and Quantification Systems Product Siz
86. s 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 Active SwabSolution Reagent carried over into the amplification reaction Larger loci are most susceptible to reagent 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 reagent 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 Use only a heat block to maintain efficient heat transfer Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD034 Printed in USA Revised 7 14 Page 54 Symptoms Causes and Comments Peak height imbalance continued 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 re freeze avoid multiple
87. speed This requires a silver or gold plated silver sample block The ramp speed is set after the thermal cycling run is started The Select Method Options screen appears Select Max for the ramp speed and enter the reaction volume 5 Instrument Setup and Sample Preparation 5 A Detection of Amplified Fragments Using the Applied Biosystems 3500 or 3500xL Genetic Analyzer We do not recommend use of this product with the POP 7 polymer due to artifacts that may migrate within the fluorescein and JOE channels 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 Biosystems Cat 4410228 POP 4 polymer for the Applied Biosystems 3500 or 3500xL Genetic Analyzer anode buffer container cathode buffer container conditioning reagent pouch for the Applied Biosystems 3500 or 3500xL Genetic Analyzer MicroAmp optical 96 well plate and septa or equivalent Hi Di formamide Applied Biosystems Cat 4311320 PowerPlex 5 Dye Matrix Standards 3100 3130 Cat DG4700 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 breakdo
88. st be run with Max mode as the ramp speed This requires a silver or gold plated silver sample block The ramp speed is set after the thermal cycling run is started The Select Method Options screen appears Select Max for the ramp speed and enter the reaction volume 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 four 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 24 27 cycles 5 Following amplification use your laboratory s validated separation and detection protocols to determine the optimal cycle number for the sample type and number of storage card punches 4 C Direct 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 react
89. t the software will call as a peak Values for peak amplitude thresholds are usually 50 150RFU and should be determined by individual laboratories Peak heights for the CC5 ILS 500 are generally lower than those for the other dyes Therefore the threshold for the orange dye may be lower than that for the other dyes Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD034 Printed in USA Revised 7 14 Page 44 9779TA Figure 21 The GeneMapper ID 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 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD034 Revised 7 14 11 Select the Peak Quality tab You may change the settings for peak quality Note For Steps 11 and 12 see the GeneMapper ID user s manual for more information 12 Select the Quality Flags tab You may change these settings 13 Select OK to save your settings Processing Data for Databasing or 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
90. t 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 storage card Take punches from a different portion of the card Increasing cycle number can improve low peak heights Too much sample in the reaction Use one or two 1 2mm storage card punches 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 Page 51 7 B Direct Amplification of DNA From Storage Card Punches continued Symptoms Causes and Comments Faint or absent allele peaks Amplification was inhibited when using more than one continued storage card punch with blood Use only one 1 2mm storage card punch with blood 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 mu
91. tab Figure 16 7 Select the bins text file that was imported in Section 6 A Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD034 Printed in USA Revised 7 14 Page 34 8 We recommend the values shown in Figure 16 for proper filtering of stutter peaks when using the PowerPlex 21 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 9 Select the Peak Detector tab Figure 14 shows an example of settings used at Promega You may need to optimize these settings In house validation should be performed Notes 1 Select full range or partial range for the analysis range When using a partial range choose an appropriate analysis range based on your data Choose a start point after the primer peak and just before the 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 3100 and 3100 Avant Genetic Analyzers and Applied Biosystems 3130 and Promega Corporation 2800 Woods Hollow
92. these settings In house validation should be performed Page 31 9777TA Figure 13 The GeneMapper ID X Allele tab 6 D Creating a Casework Analysis Method with GeneMapper ID X Software Version 1 2 continued 10 Select the Peak Detector tab Figure 14 shows an example of settings used at Promega You may need to optimize these settings In house validation should be performed Notes 1 Select full range or partial range for the analysis range When using a partial range choose an appropriate analysis range based on your data Choose a start point after the primer peak and just before the 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 for data generated on the ABI PRISM 3100 and 3100 Avant Genetic Analyzers and Applied Biosystems 3130 and 3130xl Genetic Analyzers For the Applied Biosystems 3500 and 3500xL Genetic Analyzers Life Technologies suggests an analysis threshold of 175RFU under their default injection conditions However individual laboratories should determine their peak amplitude thresholds from internal validation studies Peak heights for the CC5 ILS 500 are generally lower than Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526
93. to start analyzing data in GeneMapper ID X software They are not intended as a comprehensive guide for using GeneMapper ID X software We recommend that users contact Applied Biosystems for training on the software 1 Select Tools then GeneMapper ID X Manager 2 Select the Analysis Methods tab 3 Select New and a new analysis method dialog box will open 4 In the Analysis Method Editor window select GeneMapper ID X Security Group as the Security Group This allows access for all users of the software Other security groups may be used 5 Enter a descriptive name for the analysis method such as PowerPlex21 6 Select the Allele tab Figure 13 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 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD034 Printed in USA Revised 7 14 Page 30 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD034 Revised 7 14 9 We recommend the values shown in Figure 13 for proper filtering of stutter peaks when using the PowerPlex 21 System You may need to optimize
94. tured Not all CC5 ILS 500 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 orange channel to include peaks If peaks are low quality redefine the size standard for the sample to skip these peaks Significantly raised baseline Poor spectral calibration Perform a new spectral calibration and re run the samples Incorrect G5 spectral was active Re run samples and confirm that the PowerPlex 5 dye G5 spectral is set for G5 See instructions for instrument preparation in Section 5 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD034 Printed in USA Revised 7 14 Page 56 7 E GeneMapper ID Software Symptoms Causes and Comments Alleles not called To analyze samples with GeneMapper ID software the analysis parameters and size standard must both have Basic or Advanced as the analysis type If they are different an error is obtained To analyze samples with GeneMapper ID software at least one allelic ladder must be defined An insufficient number of CC5 ILS 500 fragments was defined Be sure to define at least
95. types using GeneMapper ID software version 3 2 We recommend that users of GeneMapper ID software version 3 2 complete the Applied Biosystems GeneMapper ID Software Human Identification Analysis Tutorial to familiarize themselves with proper operation of the software For GeneMapper ID software version 3 1 users we recommend upgrading to version 3 2 For analysis using GeneMapper ID software version 3 2 you will need the proper panels and bins text files PowerPlex_21_Panels_vX x txt and PowerPlex_21_Bins_vX x txt files where X x refers to the most recent version of the panels and bins text files Getting Started 1 To obtain the panels and bins text files for the PowerPlex 21 System go to www promega com resources tools genemapper id software panels and bin sets 2 Enter your contact information and select GeneMapper ID Select Submit 3 Save the PowerPlex_21_Panels_vX x txt and PowerPlex_21_Bins_vX x txt files where X x refers to the most recent version of the panels and bins text files to a known location on your computer Page 37 6 F PowerPlex 21 Panels and Bins Text Files with GeneMapper ID Software Version 3 2 continued 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 Panel Manager
96. un Module drop down list Lastly select G5 in the dye set drop down list Select OK 3 In the Plate Manager create a new plate record as described in the instrument user s manual In the dialog box that appears select GeneMapper Generic in the Application drop down list and select the appropriate plate type 96 well Add entries in the owner and operator windows and select OK Note If autoanalysis of sample data is desired refer to the instrument user s manual for instructions 4 In the GeneMapper plate record enter sample names in the appropriate cells Scroll to the right In the Results Group 1 column select the desired results group In the Instrument Protocol 1 column select the protocol you created in Step 2 Be sure this information is present for each row that contains a sample name Select OK Note To create a new results group select New in the drop down menu in the Results Group column Select the General tab and enter a name Select the Analysis tab and select GeneMapper Generic in the Analysis type drop down list Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD034 Printed in USA Revised 7 14 Page 26 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330
97. wn of formamide Poor quality formamide may contain ions that compete with DNA during injection which results in lower peak heights and reduced sensitivity A longer injection time may not increase the signal Formamide is an irritant and a teratogen avoid inhalation and contact with skin Read the warning label and take appropriate precautions when handling this substance Always wear gloves and safety glasses when working with formamide Sample Preparation 1 Prepare a loading cocktail by combining and mixing CC5 Internal Lane Standard 500 and Hi Di formamide as follows 2 0 l CC5 ILS 500 injections 10 0 l Hi Di formamide injections Note The volume of internal lane standard used in the loading cocktail can be increased or decreased to adjust the intensity of the size standard peaks Keep the volume of formamide at 10 0 l per well and adjust the volume added to the wells in Step 3 accordingly Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD034 Printed in USA Revised 7 14 Page 14 2 Vortex for 10 15 seconds to mix 3 Pipet 12 l of formamide internal lane standard mix into each well 4 Add 1 l of amplified sample or 1 l of PowerPlex 21 Allelic Ladder Mix Cover wells with appropriate septa Note Instrument detection limits vary therefor
98. xL Genetic Analyzer continued 7 To create a new Results Group Figure 8 navigate to the Library Select Results Group then select Create Alternatively a previously created Results Group may be used Select the Results Group Attributes according to laboratory practices Save with a descriptive name 8 To create a New Plate navigate to the Library and from the Manage menu select Plates then Create Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD034 Printed in USA Revised 7 14 Page 22 Figure 8 The Create New Results Group window 9253TA Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD034 Revised 7 14 9 Assign a descriptive plate name Select the plate type HID from the drop down menu Figure 9 10 Select Assign Plate Contents Figure 10 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 Page 23 Figure 10 Assigning plate contents 9255TA Figure 9 Defining plate properties 9254TA
99. ymerase which is a component of the PowerPlex 21 5X Master Mix This manual contains protocols for use of the PowerPlex 21 System with the GeneAmp PCR System 9700 thermal cycler 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 about other Promega fluorescent STR systems is available upon request from Promega or online at www promega com Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD034 Printed in USA Revised 7 14 Page 2 2 Product Components and Storage Conditions Product Size Cat PowerPlex 21 System 200 reactions DC8902 PowerPlex 21 System 4 200 reactions DC8942 Not For Medical Diagnostic Use Cat DC8902 contains sufficient reagents for 200 reactions and Cat DC8942 contains sufficient reagents for 800 reactions of 25 l each Each 200 reaction pack includes Pre amplification Components Box 1ml PowerPlex 21 5X Master Mix 1ml PowerPlex 21 5X Primer Pair Mix 25 l 2800M Control DNA 10ng l 5 1 250 l Water Amplification Grade Post amplification Components Box 100 l PowerPlex 21 Allelic Ladder Mix 2 300 l CC5 Internal Lane Standard 500 The PowerPlex 21 Allelic
100. 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 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 analysis method dialog box will open 4 In the Analysis Method Editor window select GeneMapper ID X Security Group as the Security Group This allows access for all users of the software Other security groups may be used 5 Enter a descriptive name for the analysis method such as PowerPlex21 20 Filter 6 Select the Allele

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