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PowerPlex® Y System

1. 50 Advantages of Using the Loci in the PowerPlex Y System eee 50 B DNA Extraction and Quantitation Methods and Automation Support 52 C Ibe nerna Lane Standard OUOU esner n ne 52 D Preparing the PowerPlex Y System PCR Amplification Mix 53 E Composition ot Butters and 5O UTIOTIs ear criteri acer uod eaaet ar 54 S 845 e etre een ee ere eer err eee eee 54 Description STR short tandem repeat loci consist of short repetitive sequence elements 3 7 base pairs in length 1 4 These repeats are well distributed throughout the human genome and are a rich source of highly polymorphic markers which may be detected using the polymerase chain reaction 5 8 Alleles of STR loci are differentiated by the number of copies of the repeat sequence contained within the amplified region and are distinguished from one another using fluorescence detection following electrophoretic separation STR markers on the Y chromosome Y STR have qualities that are distinct from autosomal markers and are useful for human identification 9 15 Y STR markers are found on the nonrecombining region of the Y chromosome NRY and produce a haploid profile when amplified from male DNA This quality simplifies male female mixture interpretation by removing the female contribution from an amplification profile 16 17 Strict paternal inheritance of these markers makes them useful for paternity and kinship studie
2. 3 Create anew GeneScan run and use the following settings Plate Check Module Plate Check A PreRun Module PR GS 36A 2400 Run Module GS 36A 2400 Collect time 2 5 hours Well to Read distance 36cm 4 Select the appropriate sample sheet and comb selection by using the drop down menus 5 Select the appropriate gel matrix file Section 3 B Gel Pre Run 1 Remove clamps from the polymerized acrylamide gel If necessary clean any excess acrylamide from the glass plates with paper towels saturated with deionized water 2 Shave any excess polyacrylamide away from the comb and remove the comb If using a sharkstooth comb carefully insert the sharkstooth comb teeth into the gel approximately 1 2mm 3 Position the gel glass plate unit in the 377 cassette 4 Secure the cassette in the instrument and perform a plate check as recommended in the ABI PRISM 377 DNA Sequencer User s Manual If the horizontal line graph is not flat remove the cassette clean the plate surface and repeat the plate check 5 Add TBE 1X buffer to the top and bottom buffer chambers of the instrument 6 Using a 60cc syringe filled with buffer remove any air bubbles from the well area of the gel and place the lid on the upper buffer chamber Using a syringe fitted with a bent 18 gauge needle remove any air bubbles from the bottom of the gel 7 Attach the heating plate connect the water tubing attach all electrodes close the instrument door and se
3. l TEE Flus Stutter Ratio Plus Stutter Distance TUE Amelogenin Cutoff Range Filter Factory Defaults 6361TA Figure 3 The Allele tab Select the bins text file Promega_Bins_ID3 2 X txt where X refers to the most recent version of the bins text file 9 Enter the values shown in Figure 3 for proper filtering of stutter peaks when using the PowerPlex Y System For an explanation of the proper usage and effects of these settings refer to the Applied Biosystems User Bulletin titled Installation Procedures and New Features for GeneMapper ID Software 3 2 Note Some of these settings have been optimized and are different from the recommended settings in the user bulletin 10 Select the Peak Detector tab We recommend the settings shown in Figure 4 Note Select full range or partial range for the analysis range When using a partial range choose an appropriate analysis range based on 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 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
4. Fax 608 277 2516 www promega com Partt TMD018 Page 40 Printed in USA Revised 5 12 The PowerPlex Y System uses the Schneider primer binding sites to allow high male specificity with a much smaller product size compared to the Kayser sites Concordance proficiency testing can be accomplished with the U S National Institute of Standards and Technology NIST Standard Reference Material SRM 2395 Human Y Chromosome DNA Profiling Standard Gaithersburg MD 7 Troubleshooting For questions not addressed here please contact your local Promega Branch Office or Distributor Contact information available at www promega com E mail genetic promega com 7 A Amplification and Fragment Detection Symptoms Faint or absent allele peaks Causes and Comments Impure template DNA Because of the small amount of template used this is rarely a problem Depending on the DNA extraction procedure used and sample source inhibitors may be present in the DNA sample Insufficient template Use the recommended amount of male template DNA Insufficient template Low copy number LCN analysis using capillary electrophoresis may benefit from reducing competing charged particles during injection This can be accomplished with post PCR cleanup or desalting lower conductivity formamide or reduced amounts of ILS 600 Perform in house validation for any of these methods Insufficient enzyme activity Use the recommended amount of AmpliTaq Gold
5. Symptoms Causes and Comments C Allelic ladder not running Allelic ladder and primer pair mix were not compatible e the same as samples Ensure that the allelic ladder is from the same system as the primer pair mix Promega Buffer incompatibility Samples were diluted in the wrong buffer Use Gold STXR 1X Buffer to dilute samples Poor quality formamide Use only Hi Di formamide when analyzing samples Be sure the allelic ladder and samples are from the same instrument run Migration of samples changed slightly over the course of a CE run with many samples This may be due to changes in temperature or the CE column over time Use a different injection of allelic ladder to determine sizes Poor injection of allelic ladder Include more than one ladder per instrument run Peak height imbalance Excessive amount of DNA Amplification of gt 1ng of male template can result in an imbalance with smaller loci showing more product than larger loci Use less template or reduce the number of cycles in the amplification program by 2 4 cycles 10 20 or 10 18 cycling to improve locus to locus balance Note Dilution of overamplified samples can result in dropout of larger loci Degraded DNA sample DNA template was degraded and larger loci show diminished yield Repurify the template DNA Insufficient male template DNA Use the recommended amount of male template DNA Stochastic effects can occur when amplifying low amounts of template
6. green arrow button to start data analysis 6 C Creating a Databasing or Paternity Analysis Method with GeneMapper ID Software Version 3 2 p qo r1 Select Tools then GeneMapper Manager Select the Analysis Methods tab Select New and a new analysis method dialog box will open Select HID and select OK Note If you do not see the HID option you do not have the GeneMapper ID software Contact Promega Technical Services by e mail genetic promega com for assistance Enter a descriptive name for the analysis method such as PowerPlexY_20 filter Select the Allele tab Select the bins text file corresponding to the PowerPlex System Promega Bins ID3 2 X where X refers to the most recent version of the bins text file 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 TMD018 Page 30 Printed in USA Revised 5 12 9 Enterthe values shown in Figure 7 for proper filtering of peaks when using C the PowerPlex Y System For an explanation of the proper usage and ke effect of these settings refer to the Applied Biosystems user bulletin titled Pre Installation Procedures and New Features for GeneMapper ID Software 3 2 dda g Analysis Method Editor HID General Allele Peak Detector
7. DG4640 crushed ice or ice water bath The quality of formamide is critical Use Hi Di formamide Freeze formamide in aliquots at 20 C Multiple freeze thaw cycles or long term storage at 4 C may cause breakdown of formamide Poor quality formamide may contain ions that compete with DNA during injection which results in lower peak heights and reduced sensitivity A longer injection time may not increase the signal Formamide is an irritant and a teratogen avoid inhalation and contact with skin Read the warning label and take appropriate precautions when handling this substance Always wear gloves and safety glasses when working with formamide Sample Preparation L Prepare a loading cocktail by combining Internal Lane Standard 600 ILS 600 and Hi Di formamide as follows 1 0u1 ILS 600 x injections 24 0ul Hi Di formamide x f injections Note The volume of internal lane standard used in the loading cocktail can be increased or decreased to adjust the intensity of the size standard peaks The optimal peak height for the 100 base fragment of the internal lane standard is 500 1 000RFU If peak heights are too high we recommend altering the loading cocktail to contain 0 5ul of ILS 600 and 24 5ul of Hi Di formamide Vortex for 10 15 seconds to mix Combine 25 0ul of prepared loading cocktail and 1 0ul of amplified sample Note Instrument detection limits vary therefore injection time injection volt
8. Miscellaneous balance problems Thaw the 10X Primer Pair Mix and Gold ST R 10X Buffer completely and vortex for 15 seconds before using Do not centrifuge the 10X Primer Pair Mix after mixing Calibrate thermal cyclers and pipettes routinely Using a 59 C annealing temperature instead of 60 C has been shown to improve balance in some instances Impure template DNA Inhibitors that may be present in forensic samples can lead to allele dropout or imbalance Presence of female DNA Female DNA at a concentration gt 100X that of the male component can decrease the relative yield of some loci Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD018 Revised 5 12 Page 43 e 7 B GeneMapper ID Analysis Software pP Symptoms Causes and Comments mega Alleles not called To analyze samples with GeneMapper ID software the analysis parameters and size standard must both have Basic or Advanced as the analysis type If they are different an error is obtained Figure 11 An insufficient number of ILS 600 fragments was defined Be sure to define at least one ILS 600 fragment smaller than the smallest sample peak or allelic ladder peak and at least one ILS 600 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 ILS 600 peaks de
9. Peak Quality Quality Flags Bin Set Promega_Bins_ID3 2 x Use markerspecific stutter ratio if available PTT TY Marker Repeat Type Cutoff Value MinusA Ratio Minuss Distance Minus Stutter Ratio Minus Stutter Distance From l To Flus Stutter Ratio Plus Stutter Distance TECC p o D B Amelogenin Cutoff joo Range Filter Factory Defaults OK Cancel Figure 7 The Allele tab with settings for using a 20 peak filter Select the bins text file Promega_Bins_ID3 2 X txt where X refers to the most recent version of the bins text file 6362TA Creating a Size Standard 1 Select Tools then GeneMapper Manager 2 Select the Size Standard tab 3 Select New 4 Select Basic or Advanced Figure 5 The type of analysis method selected must match the type of analysis method created earlier Select OK 5 Enter a detailed name such as ILS 600 advanced in the Size Standard Editor Figure 6 6 Choose Red for the Size Standard Dye 7 Enter the sizes of the internal lane standard fragments see Section 9 C Figure 13 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 Partt TMD018 Revised 5 12 Page 31 C 6 C Creating a Databasing or Paternity Analysis Method with GeneMapper ID Software
10. Version 3 2 continued Promega Processing Sample Data for Databasing or Paternity Samples 1 Import sample files into a new project as described in the Applied Biosystems GeneMapper ID Software Human Identification Analysis Tutorial 2 Inthe Sample Type column use the drop down menu to select Ladder Sample Positive Control or Negative Control Every folder in the project must contain at least one ladder that is designated as such for proper genotyping 3 Inthe Analysis Method column select the analysis method created in the Creating a Databasing or Paternity Analysis Method section 4 Inthe Panel column select PowerPlex_Y_ID3 2 X where X refers to the most recent version of the panels files This is the panels text file that was imported in Section 6 A 5 Inthe Size Standard column select the size standard that was created in the Creating a Size Standard section 6 If analyzing data from an ABI PRISM 310 Genetic Analyzer or an ABI PRISM 377 DNA Sequencer ensure that the appropriate matrix file is selected in the Matrix column 7 Select Analyze green arrow button to start the data analysis 6 D Sample Analysis Using the GeneScan Software and Windows Operating Systems 1 Analyze data using the GeneScan analysis software 2 Review the raw data for one or more sample runs Highlight the sample file name then in the Sample menu select raw data Move the cursor so the crosshair
11. Applied Biosystems 3130 and 3130x Genetic Analyzers A matrix must be generated for each individual instrument The PowerPlex Matrix Standards 310 Cat DG4640 is required for matrix standardization for the ABI PRISM 310 Genetic Analyzer and ABI PRISM 377 DNA Sequencer The PowerPlex Matrix Standards 3100 3130 Cat DG4650 cannot be used to generate a matrix on the ABI PRISM 310 Genetic Analyzer The PowerPlex Matrix Standards 3100 3130 Cat DG4650 is required for spectral calibration on the ABI PRISM 3100 and 3100 Avant Genetic Analyzers and Applied Biosystems 3130 and 3130xI Genetic Analyzers The PowerPlex Matrix Standards 310 Cat DG4640 cannot be used to generate a matrix on these instruments For protocols and additional information on matrix standardization see the PowerPlex Matrix Standards 310 Technical Bulletin TBD021 which is supplied with Cat DG4640 For protocols and additional information on spectral calibration see the PowerPlex Matrix Standards 3100 3130 Technical Bulletin TBD022 which is supplied with Cat DG4650 These manuals are available online at www promega com protocols 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 TMD018 Page 6 Printed in USA Revised 5 12 4 Protocols for DNA Amplification Using the PowerPlex Y System C Materials t
12. OK The Panel Manager window will close automatically Creating a Casework Analysis Method with GeneMapper ID Software Version 3 2 For detailed instructions see the Applied Biosystems GeneMapper ID software tutorial ge Tu qe rf Select Tools then GeneMapper Manager Select the Analysis Methods tab Select New and a new analysis method dialog box will open 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 Enter a descriptive name for the analysis method such as PowerPlexY advanced Select the Allele tab Figure 3 Select the bins text file corresponding to the PowerPlex System Promega Bins ID3 2 X where X refers to the most recent version of the bin set 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 Partt TMD018 Page 26 Printed in USA Revised 5 12 Analysis Method Editor HID EU x C General Allele Peak Detector Peak Quality Quality Flags Bin Set Promega_Bins_103 2 x C mega Use makerspecific stutter ratio if available 4 PRPPTTTTTE Marker Repeat Type Cutoff Value MinusA Ratio MinusA Distance Minus Stutter Ratio ha ATT E Minus Stutter Distance From
13. Ranger Singel pack for ABI 377 36cm Cambrex Cat 50691 e 10 Ammonium Persulfate Cat V3131 e TEMED e TBE 10X buffer e Nalgene tissue culture filter 0 2 micron e 36cm front and rear glass plates e 36cm gel spacers 0 2mm thick 36 well sharkstooth comb or 34 well square tooth comb 0 2mm thick e clamps e g large office binder clamps e gel loading pipette tips aerosol resistant pipette tips Section 9 F Liqui NoX or other detergent e lowerPlex Matrix Standards 310 Cat DG4640 e Blue Dextran Loading Solution Cat DV4351 e crushed ice or ice water bath e 95 C dry heating block water bath or thermal cycler O Acrylamide Long Ranger gel solution is a neurotoxin and suspected carcinogen avoid inhalation and contact with skin Read the warning label and take appropriate precautions when handling this substance Always wear gloves and safety glasses when working with acrylamide solutions Polyacrylamide Gel Preparation The following protocol is for preparation of a 36cm denaturing polyacrylamide gel for use with the ABI PRISM 377 DNA Sequencer Low fluorescence glass plates are recommended and may be obtained from the instrument manufacturer 1 Thoroughly clean glass plates with hot water and a 1 Liqui Nox solution Rinse extremely well using deionized water Allow the glass plates to air dry in a dust free environment 2 Assemble glass plates by placing 0 2mm side gel spacers b
14. USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Partt TMD018 Revised 5 12 Page 27 C 6 B Creating a Casework Analysis Method with GeneMapper ID Software e continued Promega Analysis Method Editor HID paana i Full Range Stat PEO Stop PI 10000 5723TA Figure 4 The Peak Detector tab Creating a Size Standard 1 2 3 4 Select Tools then GeneMapper Manager Select the Size Standard tab Select New Select Basic or Advanced Figure 5 The type of analysis method selected must match the type of analysis method created earlier Select 4 OK Enter a detailed name such as ILS 600 advanced in the Size Standard Editor Figure 6 Choose Red for the Size Standard Dye Enter the sizes of the internal lane standard fragments see Section 9 C Figure 13 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 Partt TMD018 Page 28 Printed in USA Revised 5 12 Select Dye and Analysis Method x C O Classic D ye Red Select Sample 5725TA Figure 5 The Select Dye and Analysis Method window CAT Standard Editor Edit Size Standard Description Name Description Size Standard Dwe Size Standard Table Size in Basepairs tee EP Re C r
15. a new plate record Name the plate and select GeneScan Select the plate size 96 well Select Finish Complete the plate record spreadsheet for the wells you have loaded Enter appropriate information into the Sample Name and Color Info columns For allelic ladder samples insert the word ladder into the Color Info column for the blue yellow and green dye colors This information must be entered to successfully analyze data with the PowerTyper M Y Macro Release 2 0 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Partt TMD018 Page 16 Printed in USA Revised 5 12 8 Inthe BioLIMS Project column select 3100 Project1 from the drop down C menu e 9 Inthe Dye Set column select Z from the drop down menu Promega 10 When using the ABI PRISM 3100 Data Collection Software Version 1 0 1 or 1 1 select GeneScan36 POP4PowerPlexY 3kV 11secs 2000 from the drop down menu in the Run Module 1 column 11 To collect the data without autoanalyzing select No Selection in the Analysis Module 1 column Analysis parameters can be applied after data collection and during data analysis using the GeneScan analysis software 12 Select OK This new plate record will appear in the pending plate records table on the plate setup page of the collection software 13 Place samples in the instrument and close
16. analyzer buffer may generate peaks in the blue and green dye colors Use autoclaved water change vials and wash buffer reservoir Excessive amount of DNA Amplification of gt 2ng male DNA template can result in a higher number of stutter bands Use less template DNA or reduce the number of cycles in the amplification program by 2 4 cycles 10 20 or 10 18 cycling Pull up or bleedthrough Pull up can occur when peak heights are too high or if a poor or incorrect matrix has been applied to the samples For the ABI PRISM 310 Genetic Analyzer and 377 DNA Sequencer generate a new matrix and apply it to the samples For the ABI PRISM 3100 and 3100 Avant Genetic Analyzers and Applied Biosystems 3130 and 3130xI Genetic Analyzers perform a new spectral calibration and re run the samples nstrument sensitivities can vary Optimize the injection or gel loading conditions See Section 5 Long term storage of amplified sample in formamide can result in degradation Repeat sample preparation using fresh formamide The CE polymer was beyond its expiration date or polymer was stored at room temperature for more than one week Maintain instrumentation on a daily or weekly basis as recommended by the manufacturer Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Partt TMD018 Page 42 Printed in USA Revised 5 12
17. chromosome in forensic analysis and paternity testing Int J Legal Med 110 118 24 11 Gill P et al 2001 DNA Commission of the International Society of Forensic Genetics Recommendations on forensic analysis using Y chromosome SIRs Int J Legal Med 114 305 9 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Partt TMD018 Page 48 Printed in USA Revised 5 12 12 Roewer L et al 2001 Online reference database of European Y chromosomal short tandem repeat A STR haplotypes Forensic Sci Int 118 106 13 e 13 Butler J M et al 2002 A novel multiplex for simultaneous amplification of 20 Y chromosome STR markers Forensic Sci Int 129 10 24 Promega 14 Kayser M et dl 1997 Evaluation of Y chromosomal STRs A multicenter study Int J Legal Med 110 125 33 15 Ruitberg C M Reeder D J and Butler J M 2001 STRBase A short tandem repeat DNA database for the human identity testing community Nucl Acids Res 29 320 2 16 Prinz M et al 1997 Multiplexing of Y chromosome specific STRs and performance for mixed samples Forensic Sci Int 85 209 18 17 Prinz M et al 2001 Validation and casework application of a Y chromosome specific STR multiplex Forensic Sci Int 120 177 88 18 Krenke B et al 2003 The PowerPlex Y System Profiles in DNA 6 2 7 10 19 Presley L
18. in USA Revised 5 12 Part TMD018 Page 41 7 A Amplification and Fragment Detection continued Symptoms Extra peaks visible in one or all color channels Causes and Comments Contamination with another template DNA or previously amplified DNA Cross contamination can be a problem Use aerosol resistant pipette tips and change gloves regularly Samples were not completely denatured 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 the samples in a thermal cycler set at 4 C as this may lead to artifacts due to DNA re annealing Artifacts of STR amplification Amplification of STRs sometimes generates artifacts that appear as faint peaks one repeat unit smaller than the allele Stutter band peak heights can be high if the samples are overloaded See Section 6 H for additional information on stutter and artifacts High background Load less amplification product or decrease injection time 5ee Section 5 Capillary electrophoresis 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 CE related artifacts contaminants Contaminants in the water used with the instrument or to dilute the 10X genetic
19. is on the baseline to the right of the large primer peak before the first internal lane standard peak red Use the X value number shown at the bottom left of the window for the start position in the analysis parameters 3 Therecommended analysis parameters are shown in Figure 8 4 Theanalysis parameters can be saved in the Params folder in most installations this is located at CA AppliedBio Shared Analysis VSizecallerV Params 5 Apply the stored analysis parameters file to the samples 6 Assign a new size standard Select a sample file and highlight the arrow next to size standard Select define new Assign the size standard peaks as shown in Figure 13 in Section 9 C Store the size standard in the Size Standards folder at CA AppliedBio Shared Analysis Sizecaller SizeStandards 7 Apply the size standard file to the samples then analyze the sample files See Section 6 F for additional information on the use of the PowerTyper Y Macro Release 2 0 and Genotyper software 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 TMD018 Page 32 Printed in USA Revised 5 12 Notes C v 1 Peak heights outside the linear range of the instrument may generate artifact peaks due to instrument saturation i e overloading the sample Promega Bleedthrough pull ups from one color to another may be observe
20. modify the injection time or voltage in the run module If peak heights are higher than desired samples can be diluted in Gold STXR 1X Buffer before mixing with loading cocktail The use of too much template DNA may result in uneven allele peak heights across loci For best results use less DNA template in the amplification reactions or reduce the number of cycles in the amplification program by 2 4 cycles to achieve the desired signal intensity 5 Centrifuge plate briefly to remove air bubbles from the wells 6 Denature samples at 95 C for 3 minutes then immediately chill on crushed ice or in an ice water bath for 3 minutes Denature samples just prior to loading the instrument Instrument Preparation Refer to the instrument users manual for instructions on cleaning installing the capillary array performing a spatial calibration and adding polymer Analyze samples as described in the user s manual for the ABI PRISM 3100 or 3100 Avant Genetic Analyzer with Data Collection Software Version 2 0 and the Applied Biosystems 3130 or 3130xl Genetic Analyzer with the following exceptions 1 Inthe Module Manager select New Select Regular in the Type drop down list and select HIDFragmentAnalysis36 POP4 in the Template drop down list Confirm that the injection time is 5 seconds and the injection voltage is 3kV Lengthen the run time to 2 000 seconds Give a new name to your run module and select OK Note Instru
21. original nomenclature described by Ayub et al 23 We have carefully selected primers to avoid or minimize artifacts including those associated with Tag DNA polymerase such as repeat slippage and terminal nucleotide addition Repeat slippage 26 27 sometimes called n 4 bands stutter or shadow bands is due to the loss of a repeat unit during DNA amplification somatic variation within the DNA or both The amount of this artifact observed depends primarily on the locus and the DNA sequence being replicated Terminal nucleotide addition 28 29 occurs when Tag DNA polymerase adds a nucleotide generally adenine to the 3 ends of amplified DNA fragments in a template independent manner The efficiency with which this occurs varies with different primer sequences Thus an artifact band one base shorter than expected i e missing the terminal addition is sometimes seen We have modified primer sequences and added a final extension step of 60 C for 30 minutes 30 to the amplification protocol to provide conditions for essentially complete terminal nucleotide addition when recommended amounts of DNA template are used The presence of microvariant alleles alleles differing from one another by lengths other than the repeat length complicates interpretation and assignment of alleles There appears to be a correlation between a high degree of polymorphism a tendency for microvariants and increased mutation rate 31 32 Promega Co
22. post amplification box after opening Storage Conditions Store all components except the 2800M Control DNA at 30 C to 10 C in a nonfrost free freezer Store the 2800M Control DNA at 2 to 10 C The PowerPlex Y 10X Primer Pair Mix PowerPlex Y Allelic Ladder Mix and Internal Lane Standard ILS 600 are light sensitive and must be stored in the dark We strongly recommend that pre amplification and post amplification reagents be stored and used separately with different pipettes tube racks etc Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Partt TMD018 Page 4 Printed in USA Revised 5 12 Available Separately o Product Size Cat R R O E e a E E E Promega Blue Dextran Loading Solution 3ml DV4351 PowerTyper Macros Release 2 0 1 CD ROM DG3470 For Laboratory Use Not For Medical Diagnostic Use The PowerTyper Macros Release 2 0 for use with Genotyper software are available from Promega This CD ROM contains the file PowerTyperYMacroV2 for use with the PowerPlex Y System The macros can be also downloaded at www promega com resources tools powertyper macros The proper panels and bins text files for use with GeneMapper ID software can be obtained from the Promega web site at www promega com resources tools genemapper id software panels and bin sets Matrix standards are required for init
23. save data in tables go to the Table drop down menu highlight Export to File and save the file with the desired name and location The saved file can be viewed and analyzed using Microsoft Excel PowerTable Format Sample Sample Peak Peak Peak Peak Over Low Satura Edited Edited Info Comment Category 1 2 3 i flow Signal tion Label Row Allele Table Format Sample Category Category Category Category Category Category Category Category Info Allele1 Allele2 Allele1 Allele2 Allele1 Allele2 Allele1 Allele 2 Vertical Table Format Sample Info 11 Save the analyzed data Go to the File menu and select Save as The PowerTyper Macro is a Genotyper file and can be overwritten if Save is used instead of Save as 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 TMD018 Revised 5 12 Page 37 C 6 G Controls o 1 Observe the results for the negative control Using the protocols defined in Promega this manual the negative control should be devoid of amplification products 2 Observe the results for the 2800M Control DNA Compare the 2800M Control DNA allelic repeat sizes with the locus specific allelic ladder The expected 2800M Control DNA allele designations for each locus are listed in Table 5 Section 9 A 6 H Results Representative resul
24. should be stored separately from those used following amplification PowerPlex Y Allelic Ladder Mix and Internal Lane Standard 600 Always include a negative control reaction i e no template to detect reagent contamination We highly recommend the use of gloves and aerosol resistant pipette tips e g ART tips Section 9 F 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 TMD018 Revised 5 12 Page 5 Promega 3 A Precautions continued 3 B Some reagents used in the analysis of STR products are potentially hazardous and should be handled accordingly Table 1 describes the potential hazards associated with such reagents Table 1 Hazardous Reagents Reagents for ABI PRISM 310 3100 and 3100 Avant Genetic Analyzers and Applied Biosystems 3130 and 3130xl Genetic Analyzers Hazard formamide irritant teratogen Reagents for ABI PRISM 377 DNA Sequencer Hazard acrylamide Long Ranger Gel Solution suspected carcinogen toxic ammonium persulfate oxidizer corrosive formamide contained in the Blue Dextran Loading Solution irritant teratogen TEMED corrosive flammable urea irritant Matrix Standardization or Spectral Calibration Proper generation of a matrix file is critical to evaluate multicolor systems with the ABI PRISM 310 3100 and 3100 Avant Genetic Analyzers and
25. then 60 C for 30 minutes 4 C soak Protocol for the Perkin Elmer Model 480 Thermal Cycler 95 C for 11 minutes then 96 C for 2 minutes then 94 C for 1 minute 60 C for 1 minute 70 C for 1 5 minutes for 10 cycles then 90 C for 1 minute 58 C for 1 minute 70 C for 1 5 minutes for 22 cycles then 60 C for 30 minutes 4 C soak When using the GeneAmp PCR System 9700 thermal cycler the ramp rates indicated in the cycling program must be set and the program must be run in 9600 ramp mode The ramp rates are set in the Ramp Rate Modification screen While viewing the cycling program navigate to the Ramp Rate Modification screen by selecting More then Modify On the Ramp Rate Modification screen the default rates for each step are 100 The rate under each hold step is the rate at which the temperature will change to that hold temperature Figure 2 shows the ramp rates for the GeneAmp PCR System 9700 thermal cycler The ramp mode is set after start has been selected for the thermal cycling run A Select Method Options screen appears Select 9600 ramp mode and enter the reaction volume 3 tmp 10 cycles 29 90 0 C 400 3 tmp 22 cycles 29 I l I I I i 70 09C I l l l l I I 7486MB Figure 2 The ramp rates for the GeneAmp PCR System 9700 thermal cycler Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 P
26. were selected because they represent well characterized loci generally accepted for forensic use This multiplex includes all loci in the European minimal haplotype DYS19 DYS385a b DYS3891 II DYS390 DYS391 DYS392 and DYS393 and the Scientific Working Group DNA Analysis Methods SWGDAM recommended Y STR panel European minimal haplotype plus DYS438 and DYS439 plus DYS437 More information on the European minimal haplotype can be found at www ystr org 12 The PowerPlex Y System includes an extensive allelic ladder containing the most common variants observed at each locus Table 5 lists the alleles in the allelic ladder and the haplotype of the 2800M Control DNA Table 4 The PowerPlex Y System Locus Specific Information Chromosomal GenBank Accession Repeat Sequence STR Locus Label Location Number 5293 DYS391 FL Yq G09613 TCTA 14 Ip SO MENS Yq AF140635 TCTG TCTA Complex 14 DYS439 FL Yq AC002992 GATA 23 DYS393 TMR Yq G09601 AGAT 14 DYS390 TMR Yq AC011289 TCTG TCTA Complex 14 DYS385a b TMR Yq 793950 GAAA 14 DYS438 JOE Yq AC002531 ETTIC 23 DYS437 JOE Yq AC002992 TCTA TCTG Complex 23 DYS19 JOE Yq X77751 TAGA Complex 14 DYS392 JOE Yq 09867 TAT 14 The August 1997 report 24 25 of the DNA Commission of the International Society for Forensic Haemogenetics ISFH states 1 for STR loci within coding genes the coding strand shall be used and the repeat sequence motif defined using t
27. 0 170 490 210 230 250 270 290 310 330 350 1200 1000 800 500 400 200 0 Tu A adt 44 16 amp 1oftalialielis TOP MUN OON ee NUR NE CR ELS 1200 1000 800 500 400 200 l U glie 18 0 g2 24 26 elb Hi haksiz keler 23 as E 16 12 14 16 19 1 B3 Bs 7 00 03 14 16 18 20 23 24 E Figure 10 The PowerPlex Y Allelic Ladder Mix The PowerPlex Y Allelic Ladder Mix was analyzed with an Applied Biosystems 3130 Genetic Analyzer using a 3kV 3 second injection The sample file was analyzed with GeneMapper ID software version 3 2 and PowerPlex Y panels and bins text files Panel A The fluorescein labeled allelic ladder components and their allele designations Panel B The JOE labeled allelic ladder components and their allele designations Panel C The TMR labeled allelic ladder components and their allele designations 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 TMD018 Revised 5 12 Page 39 Promega 6 H Results continued Stutter and Artifacts Stutter products are a common amplification artifact associated with STR analysis The pattern and intensity of stutter may differ between primer sets for the same loci due to reaction and amplification conditions and the labeled strand direction For samples with increased signal or template amount stutter produ
28. 01 Slicprep 96 Device 10 pack V1391 Not for Medical Diagnostic Use For Research Use Only Not for use in diagnostic procedures 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 TMD018 Revised 5 12 Page 55 C 9 F Related Products continued Y Polyacrylamide Gel Electrophoresis Reagents Promega Product Size Cat Ammonium Persulfate Molecular Grade 25g V3131 TBE Buffer 10X Molecular Biology Grade Ne V4251 Urea 1kg V3171 Blue Dextran Loading Solution 3ml DV4351 ART Aerosol Resistant Tips Product Volume Size tips pack Cat ARTS 10 Ultramicro Pipet Tip 0 5 10ul 960 DY1051 ART 20E Ultramicro Pipet Tip 0 5 10ul 960 DY1061 ART 20P Pipet Tip 20ul 960 DY1071 ART GEL Gel Loading Pipet Tip 100u1 960 DY1081 ART 100 Pipet Tip 100u1 960 DY1101 ART 100E Pipet Tip 100u1 960 DY1111 ART 200 Pipet Tip 200ul 960 DY1121 ART 1000E Pipet Tip 1 000u1 800 DY1131 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD018 Printed in USA Page 56 Revised 5 12 JSTR loci are the subject of U S Pat No RE 37 984 German Pat No DE 38 34 636 C2 and other patents issued to the Max Planck Gesellschaft zur F rderung der Wissenschaften e V Germany The purchase of this p
29. 26 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD018 Printed in USA Page 14 Revised 5 12 5 B Detection of Amplified Fragments Using the ABI PRISM 3100 Genetic C Analyzer with Data Collection Software Version 1 0 1 or 1 1 ke Materials to Be Supplied by the User Promega e 95 C dry heating block water bath or thermal cycler e crushed ice or ice water bath aerosol resistant pipette tips e 3100 capillary array 36cm e performance optimized polymer 4 POP 4 for the 3100 e 10X genetic analyzer buffer with EDTA e MicroAmp optical 96 well plate and septa for the 3100 Hi Di formamide Applied Biosystems Cat 4311320 e lowerPlex Matrix Standards 3100 3130 Cat DG4650 O 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 O Formamide is an irritant and a teratogen avoid inhalation and contact with skin Read the warning label and take appropriate precautions when handling this substance Always wear gloves and safety glasses when working with formamide Sample Preparation 1 Prepare a loading cocktail by combining and mixing Internal Lane Standard 600 and Hi Di
30. 7 2516 www promega com Printed in USA Part TMD018 Revised 5 12 Page 47 7 C PowerTyper Y Macro continued Symptoms Causes and Comments The plots window or allele The macros were not run in the proper order Use the POWER table does not display all data or POWER 20 Filter macro option All four dye colors were not imported For Genotyper software versions 2 5 and 3 5 or higher set preferences in the Edit menu to import the blue green yellow and red colors The Check ILS macro All four dye colors were not imported For Genotyper displays an empty plot software versions 2 5 and 3 5 or higher set preferences in the window Edit menu to import the blue green yellow and red colors Off ladder peaks Migration of samples changed slightly over the course of a CE run with many samples This may be due to changes in temperature or the CE column over time Use a different injection of allelic ladder to determine sizes in the PowerTyper Y Macro Release 2 0 Do not use the first injection on a new column for the ladder sample The base pair size of alleles was incorrect because incorrect fragment sizes were assigned to the internal lane standard Confirm that internal lane standard fragments are assigned correctly Re analyze the sample using GeneScan software and redefine the internal lane standard fragments 8 References 1 Edwards A et al 1991 DNA typing with trimeric and tetrameric tandem repeats Polymorphic
31. 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 20 Hartmann J M et al 1991 Guidelines for a quality assurance program for DNA analysis Crime Laboratory Digest 18 44 75 21 F redi S et al 1999 Y STR haplotyping in two Hungarian populations Int J Legal Med 113 38 42 22 Schneider P M et al 1998 Tandem repeat structure of the duplicated Y chromosomal STR locus DYS385 and frequency studies in the German and three Asian populations Forensic Sci Int 97 61 70 23 Ayub Q et al 2000 Identification and characterisation of novel human Y chromosomal microsatellites from sequence database information Nucl Acids Res 28 e8 24 B r W et al 1997 DNA recommendations Further report of the DNA Commission of the ISFH regarding the use of short tandem repeat systems Int J Legal Med 110 175 6 25 Gill P et al 1997 Considerations from the European DNA Profiling Group EDNAP concerning STR nomenclature Forensic Sci Int 87 185 92 26 Levinson G and Gutman G A 1987 Slipped strand mispairing A major mechanism for DNA sequence evolution Mol Biol Evol 4 203 21 27 Schlotterer C and Tautz D 1992 Slippage synthesis of simple sequence DNA Nucl Acids Res 20 211 5 28 Smith J R et al 1995 Approach to genotyping erro
32. DNA polymerase The template DNA will be added at Step 6 Assumes the AmpliTaq Gold DNA polymerase is at 5u ul If the enzyme concentration is different the volume of enzyme must be adjusted accordingly Store DNA templates in nuclease free water or TE buffer 10mM Tris HCl pH 8 0 0 1mM EDTA If the DNA template is stored in TE buffer that is not pH 8 0 or contains a higher EDTA concentration the volume of DNA added should not exceed 20 of the final reaction volume Amplification efficiency and quality can be 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 8 For the negative amplification control pipet nuclease free water or TE buffer instead of template DNA into a reaction tube containing PCR amplification mix 9 If using the GeneAmp PCR System 9600 9700 or 2400 thermal cycler and MicroAmp reaction tubes or plates no addition of mineral oil to the reaction tubes is required However if using the model 480 thermal cycler and GeneAmp reaction tubes add one drop of mineral oil to each tube before closing Note Allow the mineral oil to flow down the side of the tube and form an overlay to limit sample loss or cross contamination due to splattering 10 Optional Briefly centrifuge the tubes to bring cont
33. DNA polymerase Check the expiration date on the tube label Incorrect amplification program Confirm the program High salt concentration or altered pH If the DNA template is stored in TE buffer that is not pH 8 0 or contains a higher EDTA concentration the DNA volume should not exceed 20 of the total reaction volume Carryover of K Na Mg or EDTA from the DNA sample can negatively affect PCR A change in pH also may affect PCR Store DNA in TE buffer 10mM Tris HCl pH 8 0 0 1mM EDTA or nuclease free water Thermal cycler plate or tube problems Review the thermal cycling protocols in Section 4 B We have not tested other reaction tubes plates or thermal cyclers Calibrate the thermal cycler heating block if necessary Primer concentration was too low Use the recommended primer concentration Vortex the PowerPlex Y 10X Primer Pair for 15 seconds to mix before use Heat denature samples for the recommended time and cool on crushed ice or in an ice water bath immediately prior to capillary electrophoresis Poor capillary electrophoresis injection ILS 600 peaks also affected Re inject the sample Check the syringe for leakage Check the laser power Poor quality formamide was used Use only Hi Di formamide when analyzing samples Promega 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
34. Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD018 Page 54 Printed in USA Revised 5 12 Fluorescent STR Multiplex Systems continued C Product Size Cat PowerPlex ESX 16 System 100 reactions DC6711 Promega 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 55 System 100 reactions DC6951 400 reactions DC6950 Not for Medical Diagnostic Use Accessory Components Product Size Cat PowerPlex Matrix Standards 310 50ul each dye DG4640 PowerPlex Matrix Standards 3100 3130 25ul each dye DG4650 2800M Control DNA 25ul DD7101 500ul DD7251 9948 Male DNA 250ng DD2061 9947A DNA 250ng DD1001 Internal Lane Standard 600 150ul DG1071 Gold ST R 10X Buffer 1 2ml DM2411 Mineral Oil 12ml DY1151 Water Amplification Grade 6 250ul 5 x 1 250yl DW0991 Not for Medical Diagnostic Use Sample Preparation Systems Product Size Cat DNA IQ System 100 reactions DC6701 400 reactions DC6700 Casework Extraction Kit 100 reactions DC6745 Differex System 20 samples DC6801 200 samples DC6800 Maxwell 16 Forensic Instrument each AS3060 DNA IQ Reference Sample Kit for Maxwell 16 48 preps AS1040 DNA IQ Casework Pro Kit for Maxwell 16 48 preps AS1240 Plexor HY System 800 reactions DC1000 200 reactions DC10
35. Technical Manual PowerPlex Y System INSTRUCTIONS FOR USE OF PRODUCTS DC6760 AND DC6761 PRINTED IN USA Revised 5 12 Partt TMD018 PowerPlex Y System O All technical literature is available on the Internet at 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 IERI ed 0 EE mE 2 2 Product Components and Storage Conditions sss 4 OMM Duc B Puunme enin iSS ESE 5 A O E E EA E E erat B B Matrix Standardization or Spectral Calibration sss 6 4 Protocols for DNA Amplification Using the PowerPlex Y System 7 Ae AMPON Oe eieiaeo ae ara a a reisa n PEER 7 B Amphicaion Thermal C OTHO ose oarsestatees ettet Die optet teo ted 10 5 Instrument Setup and Sample Preparation sse 12 A 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 3130x Genetic Analyzer with Data Collection Software Version 3 0 sess 12 B Detection of Amplified Fragments Using the ABI PRISM 3100 Genetic Analyzer with Data Collection Software Version 1 0 1 or 1 1 15 C Detect
36. age or the amount of product mixed with loading cocktail may need to be adjusted If peak heights are higher than desired samples can be diluted in Gold STXR 1X Buffer before mixing with loading cocktail The use of too much template DNA may result in uneven allele peak heights across loci For best results use less template DNA in the amplification reactions or reduce the number of cycles in the amplification program by 2 4 cycles i e 10 18 or 10 20 cycling Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Partt TMD018 Page 18 Printed in USA Revised 5 12 4 Combine 25 0ul of prepared loading cocktail and 1 0ul of PowerPlex Y C Allelic Ladder Mix xe Centrifuge tubes briefly to remove air bubbles from the wells Promega 6 Denature samples and ladder by heating at 95 C for 3 minutes and immediately chill on crushed ice or in an ice water bath for 3 minutes Denature samples just prior to loading 7 Placethe tubes in the appropriate autosampler tray 8 Place the autosampler tray in the instrument and close the instrument doors Instrument Preparation Refer to the instrument users manual for instructions on cleaning the pump block installing the capillary calibrating the autosampler and adding polymer to the syringe 1 Open the ABI PRISM 310 Data Collection Software 2 Prepare a GeneScan sam
37. air Mix after vortexing as this may cause the primers to be concentrated at the bottom of the tube 2 A precipitate may form in the Gold STXR 10X Buffer If this occurs warm the solution briefly at 37 C then vortex until the precipitate is in solution 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 Place one clean 0 2ml or 0 5ml reaction tube for each reaction into a rack and label appropriately Alternatively use a MicroAmp plate and label appropriately Note If using the GeneAmp PCR System 9600 9700 or 2400 thermal cyclers use 0 2ml MicroAmp 8 strip reaction tubes or MicroAmp plate For the Perkin Elmer model 480 we recommend standard 0 5ml GeneAmp thin walled reaction tubes Add the final volume of each reagent listed in Table 2 into a sterile tube Mix gently Table 2 shows the component volumes per reaction A worksheet to calculate the required amount of each PCR amplification mix component is provided in Section 9 D Table 6 Amplification of gt 1ng of male DNA template results in an imbalance in peak heights from locus to locus The smaller loci show greater amplif
38. alysis of samples and the following error message appears when data are displayed Some selected sample s do not contain analysis data Those sample s will not be shown ABI PRISM 310 Genetic Analyzer no data will be displayed Apply a matrix file during analysis in the GeneMapper ID software and re analyze There was a conflict between different sets of panels and bins Error message after attempting to import panels and bins text files Unable to save panel data java SQLEException ORA 00001 unique constraint IFA CKP NNN violated Allelic ladder peaks are labeled off ladder OL text files Check to be sure that the bins are installed properly If not delete all panels and bins text files and re import files in a different order GeneMapper ID software was not used or microsatellite analysis settings were used instead of HID analysis settings GeneMapper software does not use the same algorithms as GeneMapper ID software and cannot correct for sizing differences using the allelic ladder Promega recommends using GeneMapper ID software for analysis of PowerPlex reactions If using GeneMapper ID software version 3 2 be sure that the analysis method selected is an HID method This can be verified by opening the analysis method using the GeneMapper Manager then selecting the General tab The analysis type cannot be changed If the method is not HID it should be deleted and a new analysis method
39. amples at 4 C or higher may produce degradation products Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD018 Printed in USA Page 10 Revised 5 12 Protocol for the GeneAmp PCR System 9700 Thermal Cycler 95 C for 11 minutes then 96 C for 1 minute then ramp 100 to 94 C for 30 seconds ramp 29 to 60 C for 30 seconds ramp 23 to 70 C for 45 seconds for 10 cycles then ramp 100 to 90 C for 30 seconds ramp 29 to 58 C for 30 seconds ramp 23 to 70 C for 45 seconds for 22 cycles then 60 C for 30 minutes 4 C soak Protocol for the GeneAmp PCR System 9600 Thermal Cycler 95 C for 11 minutes then 96 C for 1 minute then 94 C for 30 seconds ramp 68 seconds to 60 C hold for 30 seconds ramp 50 seconds to 70 C hold for 45 seconds for 10 cycles then 90 C for 30 seconds ramp 60 seconds to 58 C hold for 30 seconds ramp 50 seconds to 70 C hold for 45 seconds for 22 cycles then 60 C for 30 minutes 4 C soak Protocol for the GeneAmp PCR System 2400 Thermal Cycler 95 C for 11 minutes then 96 C for 1 minute then ramp 100 to 94 C for 30 seconds ramp 100 to 60 C for 30 seconds ramp 23 to 70 C for 45 seconds for 10 cycles then ramp 100 to 90 C for 30 seconds ramp 100 to 58 C for 30 seconds ramp 23 to 70 C for 45 seconds for 22 cycles
40. are and Versions 3 1 and 3 2 Windows Operating Macintosh Operating Systems Systems Figure 1 An overview of the PowerPlex Y System protocol 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 TMD018 Revised 5 12 Page 3 Product Components and Storage Conditions Product Size Cat PowerPlex Y System 50 reactions DC6761 Not For Medical Diagnostic Use Cat DC6761 contains sufficient reagents for 50 reactions of 25ul each Includes Pre amplification Components Box Blue Label 1 x 300ul Gold ST R 10X Buffer 1 x 125ul PowerPlex Y 10X Primer Pair Mix 25ul 2800M Control DNA 10ng ul Post amplification Components Box Beige Label 1 x 12 5ul PowerPlex Y Allelic Ladder Mix 1 x 150ul Internal Lane Standard 600 Product Size Cat PowerPlex Y System 200 reactions DC6700 Not For Medical Diagnostic Use Cat DC6760 contains sufficient reagents for 200 reactions of 25ul each Includes Pre amplification Components Box Blue Label 2 x 300ul Gold ST R 10X Buffer 4 x 125ul PowerPlex Y 10X Primer Pair Mix 25ul 2800M Control DNA 10ng ul Post amplification Components Box Beige Label 4 x 12 5ul PowerPlex Y Allelic Ladder Mix 2 x 150ul Internal Lane Standard 600 The PowerPlex Y Allelic Ladder Mix is provided in a separate sealed bag for shipping This component should be moved to the
41. art position in the analysis parameters The recommended analysis parameters are Analysis Range Start Defined in Step 2 stop 10 000 Data Processing Baseline Checked Multicomponent Checked Smooth Options Light Peak Detection Peak Amplitude Thresholds B Ax G R Min Peak Half Width 2pts Size Call Range Min 60 Max 600 Size Calling Method Local Southern Method Split Peak Correction None Smooth options should be determined by individual laboratories 7 he peak amplitude thresholds are the minimum peak heights at which the software will call a peak Values for the peak amplitude thresholds are usually 50 200RFU and should be determined by individual laboratories The analysis parameters can be saved in the Params folder Apply the stored analysis parameters file to the samples Assign a new size standard Select a sample file highlight the arrow next to size standard then select define new Assign the size standard peaks as shown in Figure 13 in Section 9 C Store the size standard in the Size Standards folder Apply the size standard file to the samples then analyze the sample files See Section 6 F for additional information on the use of the PowerTyper 16 Macro Release 2 0 and Genotyper software For additional information regarding the GeneScan analysis software refer to the GeneScan Analysis Software User s Manual Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Fr
42. created Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Partt TMD018 Page 46 Printed in USA Revised 5 12 7 C PowerTyper Y Macro C 2 Symptoms Causes and Comments File does not open Genotyper software was not installed Be certain that the Promega on your computer Genotyper software version 2 5 Macintosh or version 3 6 or higher Windows NT is installed Incorrect version of Genotyper software The PowerTyper Y Macro will not work with Genotyper software versions prior to version 2 5 Error message Allelic ladder sample files were not identified Be certain the Could not complete the Sample Info or Color Info column for each lane containing Run Macro command because PowerPlex Y Allelic Ladder Mix contains the word ladder no dye lanes are selected The macro uses the word ladder to identify the sample files containing allelic ladder All four dye colors were not imported For Genotyper software versions 2 5 and 3 5 or higher set preferences in the Edit menu to import the blue green yellow and red colors Error message Peak heights for one or more alleles in the allelic ladder Could not complete the sample file were below 150RFU The allelic ladder categories Run Macro command are defined as having a minimum peak height of 150RFU If because the labeled p
43. cts which are one and occasionally two repeat units Table 4 smaller than the true allele peak are often observed In addition to stutter peaks several other stutter like peaks can be observed at some PowerPlex Y loci The DYS392 locus and occasionally other loci with high signal or template amount may show a peak one repeat unit larger than the true allele DYS19 and DYS389II can display low level products in the n 2 and n 2 positions two bases below and above the true allele peak respectively with the DYS19 n 2 product being the most prominent DYS437 and DYS385 also may show low level peaks in the n 5 position with DYS437 DYS385 and DYS393 also displaying an n 9 to n 10 product The intensity of stutter and stutter like peaks is directly related to signal intensity Results may vary based on laboratory optimization Internal laboratory validation should be performed A low level artifact in the DYS438 region of the JOE channel may be observed between 114 120bp This artifact is not template derived and may appear in the negative control and in low product yield analyses The peak height of this artifact may increase with longer injection time or higher injection voltage In addition low level artifacts in the noncalling region between the DYS393 and DYS390 assay ranges of the TMR channel may be observed at approximately 150 165bp These artifacts also are not template derived and may appear in the negative control and in low product yi
44. d Saturated signal may appear also as two peak split peak 2 If peak heights are not within the linear range of detection of the instrument the ratio of stutter peaks to real allele peaks increases and allele designations become difficult to interpret The balance of peak heights also may appear less uniform 3 There may be variation between instruments regarding the relative fluorescent units detected using the same sample Furthermore different instruments vary in the relative efficiency of color detection affecting the dye color to dye color balance Analysis Parameters 6054TA Figure 8 The Analysis Parameters window The start point of the analysis range which will vary is defined in Section 6 D Step 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 Partt TMD018 Revised 5 12 Page 05 Promega 6 E Sample Analysis Using the GeneScan Software and Macintosh Operating Systems 1 Z Analyze data using the GeneScan analysis software Review the raw data for one or more sample runs Highlight the sample file name then in the Sample menu select raw data Move the cursor so the crosshair is on the baseline to the right of the large primer peak before the first internal lane standard peak red Use the X value number shown at the bottom left of the window for the st
45. d bin sets 2 Enter your contact information and select GeneMapper ID Select Submit 3 Select the PowerPlex Panels amp Bin Sets link and save the zip file to your computer 4 Open the files using the Windows WinZip program and save the unzipped files to a known location on your computer 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 TMD018 Revised 5 12 Page 25 Promega 6 A PowerPlex Panels and Bins Text Files with GeneMapper ID Software Version 3 2 continued 6 B Importing Panels and Bins Text Files For detailed instructions see the Applied Biosystems GeneMapper ID software tutorial Br de SS ES Open the GeneMapper ID software version 3 2 Select Tools then Panel Manager Highlight the Panel Manager icon in the upper left navigation pane Select File then Import Panels Navigate to the saved panels and bins text files Select Promega Panels ID3 2 X txt where X refers to the most recent version of the panels and bins text files Select Import In the navigation pane highlight the Promega Panels ID3 2 X folder that you just imported Select File then Import Bin Set Navigate to the saved panels and bins text files Select Promega Bins ID3 2 X txt then Import At the bottom of the Panel Manager window select
46. eak peak heights of ladder alleles are below 150RFU the software could not be found will not be able to locate the allele peak Re run the allelic ladder using more sample or longer injection time to assure peak heights above 150RFU CE spikes in the allelic ladder sample were identified as alleles by the macro Use a different injection of allelic ladder Allelic ladder data were not compatible with the PowerTyper file used Confirm that the PowerTyper Macro file matches the allelic ladder being used The base pair size of alleles in the allelic ladder were outside of the defined category range Be sure internal lane standard fragments are correctly sized Redefine internal lane standard fragments and re analyze the sample using GeneScan software Compare the size of the smallest allele in the allelic ladder with the base pair size and range listed in the categories for the same alleles If necessary increase the category start range in the category window to greater than t bp and save the macro under a new name Allelic ladder peaks were too high causing stutter peaks to be called as allele peaks Use a shorter injection time decrease the amount of allelic ladder used or re analyze the allelic ladder sample using increased peak amplitude thresholds in the GeneScan analysis parameters Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 27
47. ee in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD018 Page 34 Printed in USA Revised 5 12 Notes C 1 Peak heights outside the linear range of the instrument may generate artifact peaks due to instrument saturation i e overloading the sample Promega Bleedthrough pull ups from one color to another may be observed Saturated signal may appear also as two peaks split peak 2 If peak heights are not within the linear range of detection of the instrument the ratio of stutter peaks to real allele peaks increases and allele designations become difficult to interpret The balance of peak heights also may appear less uniform 3 There may be variation between instruments regarding the relative fluorescent levels detected using the same sample Furthermore different instruments vary in the relative efficiency of color detection affecting the dye color to dye color balance 6 F Sample Analysis Using the Genotyper Software and PowerTyper Y Macro To facilitate analysis of data generated with the PowerPlex9 Y System we have created a file to allow automatic assignment of genotypes using the Genotyper software After samples are amplified detected using the ABI PRISM 310 or 3100 Genetic Analyzer using Data Collection Software Version 1 0 1 or 1 1 or ABI PRISM 377 DNA Sequencer and analyzed using the GeneScan analysis software sample files can be imported into the Genotyper pr
48. eld analyses In amplified samples noise below the allele calling assay range in the blue lt 70bp green lt 90bp and yellow lt 95bp channels can be observed In the allelic ladder low level artifact peaks can be observed most notable is a series of peaks above DYS389II Figure 10 In general none of these artifacts should affect interpretation due to the peak intensity and position relative to the allelic ladder peaks However their intensity can be decreased by reducing the injection time or loading volume or signal thresholds can be increased during analysis to exceed the observed noise level DYS385a b Concordance Documentation of nonconcordance has been previously described for the DYS385a b locus An initial publication of primer sequences by Kayser 14 incorporates a single base deletion between the primer binding sites but outside of the repeat region 11 13 21 An alternative set of primer sequences published later by Schneider 22 produces a much smaller amplicon and is internal to this mutation site Amplification of a sample with this rare single base deletion in the DYS385a b flanking region using the Kayser primers will consistently produce an amplicon that types one base shorter in length than that generated with the Schneider sequences The Schneider primer sequences are commonly used 13 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330
49. ents to the bottom and remove any air bubbles Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Partt TMD018 Revised 5 12 Page 9 CQ 4 B Amplification Thermal Cycling Y This manual contains protocols for use of the PowerPlex Y System with the Promega Perkin Elmer model 480 and GeneAmp PCR system 9600 9700 and 2400 thermal cyclers For information on other thermal cyclers please contact Promega Technical Services by e mail genetic promega com Amplification and detection instrumentation may vary You may need to optimize protocols including cycle number and injection conditions or loading volume for each laboratory instrument Testing at Promega Corporation shows that 10 22 cycles work well for 0 5 1ng of purified DNA templates For higher amounts of input DNA or to decrease sensitivity fewer cycles such as 10 16 10 18 or 10 20 should be evaluated In house validation should be performed 1 Place the tubes or MicroAmp plate in a thermal cycler 2 Select and run a recommended protocol The preferred protocols for use with the GeneAmp PCR System 9600 9700 and 2400 thermal cyclers and Perkin Elmer model 480 thermal cycler are provided below 3 After completion of the thermal cycling protocol store the amplified samples at 20 C in a light protected box Note Long term storage of amplified s
50. etween the front and rear glass plates Hold the plates together using binder clamps 4 clamps on each side Place the assembly horizontally on a test tube rack or similar support 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 TMD018 Revised 5 12 Page 21 Promega 5 D Detection of Amplified Fragments Using the ABI PRISM 377 DNA Sequencer continued p 10 Prepare a 5 Long Ranger acrylamide gel total of 50ml by combining the ingredients listed in Table 3 Stir the solution until the urea has dissolved Table 3 Preparation of a 5 Long Ranger Polyacrylamide Gel Component 5 Gel Final Concentration urea 18g 6M deionized water 26ml 10X TBE 5ml 1X 50 Long Ranger gel solution 5ml 5 total volume 50ml Note Long Ranger Singel Packs may be used Filter the acrylamide solution through a 0 2 micron filter e g Nalgene tissue culture filter and degas for 5 minutes Add 35ul of TEMED and 250ul of fresh 10 ammonium persulfate to 50ml of acrylamide solution and mix gently Using a disposable 60cc syringe pour the gel by starting at the well end of the plates and carefully injecting the acrylamide between the horizontal glass plates Allow the solution to fill the top width of the plates While maintaining a constant flow of solution gently tap the glass plates
51. fined 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 B or 6 C Panels text file selected for analysis was incorrect for the STR system used Assign correct panels 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 Eric default LUE EnS E STUL E tate sing Info Raw Data EPT Data Sample Information gt Sample File CRE_1 2h_H0G_2004 06 L17 fsa Sample rigin Path G Ferivate Technical Service GI datai genetica CRE 172h HOS 2004 06 17 fsa Status Message Changed size standard from 1ls80500adv to ILS600 Classic File Source Disk media Error Messa
52. formamide as follows 0 5u1 ILS 600 x injections 9 5ul Hi Di formamide x injections Note The volume of internal lane standard used in the loading cocktail can be increased or decreased to adjust the intensity of the size standard peaks The optimal peak height for the 100 base fragment of the internal lane standard is 500 1 000RFU If peak heights are too low we recommend altering the formamide internal lane standard mix to contain 1 0ul of ILS 600 and 9 0ul of Hi Di formamide If peak heights are too high we recommend altering the loading cocktail to contain 0 25ul of ILS 600 and 9 75ul of formamide 2 Vortex for 10 15 seconds to mix 3 Pipet 10ul of formamide internal lane standard mix into each well Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD018 Revised 5 12 Page 15 C 5 B Detection of Amplified Fragments Using the ABI PRISM 3100 Genetic e Analyzer with Data Collection Software Version 1 0 1 or 1 1 continued Promega Add 1ul of amplified sample or 1ul of PowerPlex Y Allelic Ladder Mix Cover wells with appropriate septa Note Instrument detection limits vary therefore injection time injection voltage or the amount of product mixed with loading cocktail may need to be adjusted Use the Module Editor in the Tools menu to modify injection time
53. ge Current Settings Sample Type Sample Figure 11 The error message that appears in the GeneMapper ID software when the analysis parameters and size standard have different analysis types Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD018 Printed in USA Page 44 Revised 5 12 Symptoms Causes and Comments C Size standard not called Starting data point was incorrect for the partial range chosen 7 correctly Figure 12 in Section 6 B Adjust the starting data point in the analysis method Alternatively use a full range for the analysis Promega Extra peaks in advanced mode size standard Open the Size Match Editor Highlight the extra peak select Edit and select hi delete size label Select auto adjust sizes Run was too short and larger peaks in ILS were not captured Not all ILS 600 peaks defined in the size standard were detected during the run e Create a new size standard using the internal lane standard fragments present in the sample Re run samples using a longer run time Peaks in size standard missing If peaks are below threshold decrease the peak amplitude threshold in the analysis method for the red channel to include peaks If peaks are low quality redefine the size standard for the sample to skip these peaks Error message The size standard and analysis me
54. he 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 TMR carboxy tetramethylrhodamine FL fluorescein JOE 6 carboxy 47 5 dichloro 27 7 dimethoxyfluorescein 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 TMD018 Page 50 Printed in USA Revised 5 12 9 A Advantages of Using the Loci in the PowerPlex Y System continued C 2 Table 5 The PowerPlex Y System Allelic Ladder Information Promega Size Range of Allelic Repeat Numbers of Alleles Observed in Ladder Components Allelic Ladder 2800M Control STR Locus Label bases Components DNA DYS391 FL 90 118 6 8 13 10 DYS389I FL 148 168 10 15 14 DYS439 FL 203 231 8 152 12 DYS389 II FL 256 296 24 34 9l DYS399 TMR 104 136 8 16 13 DYS390 TMR 191 227 18 27 24 DYS385 TMR 243 315 7 25 13 16 DYS438 JOE 101 121 8 12 9 DYS437 JOE 183 199 13 17 14 DYS19 JOE 232 268 10 19 14 DYS392 JOE 294 327 7 18 13 When using an internal lane standard such as the Internal Lane Standard 600 the calculated sizes of allelic ladder components may differ from those listed This occurs because different sequences in allelic ladder and ILS components may cause differences in migration The dye label also affects migration of alleles Follows the
55. he number of cycles in the amplification program by 2 4 cycles i e 10 18 or 10 20 cycling Combine 2 0ul of prepared loading cocktail and 1 0ul of PowerPlex Y Allelic Ladder Mix Vortex the allelic ladder mix prior to pipetting Briefly centrifuge samples to bring the contents to the bottom of the tubes Just prior to loading the gel denature samples by heating at 95 C for 3 minutes and immediately chill on crushed ice or in an ice water bath Denature samples just prior to loading the gel After the 15 to 20 minute pre run pause the instrument by selecting Pause By pausing the pre run the water will continue to circulate keeping the gel warm during sample loading Use a 60cc syringe filled with buffer and fitted with a bent 18 gauge needle to flush urea from the well area Load 1 5yl of each denatured sample into the respective wells You may need to optimize the volume of sample loaded for individual instruments We recommend loading volumes of 1 0 2 0ul 10 Place the lid on the upper buffer chamber and close the instrument door Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Partt TMD018 Page 24 Printed in USA Revised 5 12 Gel Electrophoresis and Detection C 1 After loading select Cancel to stop the pre run Make sure that the run time is set at 2 5 hours then select Run to begin elec
56. hone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Revised 5 12 Part TMD018 Page 11 QO 5 Instrument Setup and Sample Preparation Y P 5 A Detection of Amplified Fragments Using the ABI PRISM 3100 or 3100 Avant mega Genetic Analyzer with Data Collection Software Version 2 0 or the Applied Biosystems 3130 or 3130x Genetic Analyzer with Data Collection Software Version 3 0 Materials to Be Supplied by the User e 95 C dry heating block water bath or thermal cycler e crushed ice or ice water bath e aerosol resistant pipette tips e 3100 or 3130 capillary array 36cm e performance optimized polymer 4 POP 4 for the 3100 or 3130 e 10X genetic analyzer buffer with EDTA e MicroAmp optical 96 well plate and septa e Hi Di formamide Applied Biosystems Cat 4311320 e PowerPlex Matrix Standards 3100 3130 Cat DG4650 Q 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 O 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 safet
57. ial setup of the color separation matrix The matrix standards are sold separately and are available for the ABI PRISM 310 Genetic Analyzer and 377 DNA Sequencer PowerPlex Matrix Standards 310 and the ABI PRISM 3100 and 3100 Avant Genetic Analyzers and Applied Biosystems 3130 and 3130xI Genetic Analyzers PowerPlex Matrix Standards 3100 3130 See Section 9 F for ordering information 3 Before You Begin 3 A Precautions The application of PCR based typing for forensic or paternity casework requires validation studies and quality control measures that are not contained in this manual 19 20 The quality of the purified DNA small changes in buffers ionic strength primer concentrations choice of thermal cycler and thermal cycling conditions can affect PCR success We suggest strict adherence to recommended procedures for amplification as well as denaturing gel electrophoresis and fluorescence detection Additional research and validation are required if any modifications are made to the recommended protocols PCR based STR analysis is subject to contamination by very small amounts of human DNA Extreme care should be taken to avoid cross contamination when preparing sample DNA handling primer pairs assembling amplification reactions and analyzing amplification products Reagents and materials used prior to amplification Gold STXR 10X Buffer 2800M Control DNA and PowerPlex Y 10X Primer Pair Mix are provided in a separate box and
58. ication yield than the larger loci Reducing the number of cycles in the amplification program by 2 to 4 cycles i e 10 20 or 10 18 cycling can improve locus to locus balance Pipet PCR amplification mix into each reaction tube Pipet the template DNA 0 5 1ng for each sample into the respective tube containing PCR amplification mix 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 Pipet 0 5ng of the diluted DNA into a reaction tube containing PCR amplification mix Note To store diluted 2800M Control DNA dilute the DNA to 0 5ng yl in TE buffer with 20pg ml glycogen and store at 4 C Do not store dilutions performed in water Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD018 Page 8 Printed in USA Revised 5 12 Table 2 PCR Amplification Mix for the PowerPlex Y System QO PCR Amplification Mix Component Volume Per Reaction nuclease free water to a final volume of 25 011 Promega Gold ST R 10X Buffer 2 5ul PowerPlex Y 10X Primer Pair Mix 2 5yl AmpliTaq Gold DNA polymerase 0 55ul 2 75u template DNA up to 1ng up to 19 45yl total reaction volume 25ul Add nuclease free water to the PCR amplification mix first then add Gold STXR 10X Buffer PowerPlex Y 10X Primer Pair Mix and AmpliTaq Gold
59. inted in USA Revised 5 12 9 D Preparing the PowerPlex Y System PCR Amplification Mix C A worksheet to calculate the required amount of each PCR amplification mix component is provided in Table 6 Multiply the volume ul per reaction by the Pro mega total number of reactions to obtain the final PCR amplification mix volume ul Table 6 PCR Amplification Mix for the PowerPlex Y System PCR Amplification Mix Volume Per Numberof _ Final Volume Component Reaction Reactions ul Gold ST R 10X Buffer 2 9 x PowerPlex Y 10X Primer Pair Mix 2 5 x AmpliTaq Gold DNA polymerase 0 55ul 2 75u x nuclease free water ul x Per tube x template DNA volume 0 25 1ng up to 19 45ul x total reaction volume 25ul x Assumes the AmpliTaq Gold DNA polymerase is at 5u ul If the enzyme concentration is different the volume of enzyme must be adjusted accordingly The amplification mix volume and template DNA volume should total 25ul Consider the volume of template DNA and add nuclease free water to the amplification mix to bring the final volume of the final reaction to 25ul 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 TMD018 Revised 5 12 Page 53 Promega 9 E Composition of Buffers and Solutions 10 ammonium persulfate TE buffer 10mM Tris HCl Add 0 05g of am
60. ion of Amplified Fragments Using the ABI PRISM 310 Selle CR ga b 71 D 18 D Detection of Amplified Fragments Using the ABI PRISM 377 DNA Pe MS INC T 21 ONE Dala So e O 25 A PowerPlex Panels and Bins Text Files with GeneMiapper ID Soliware Versiono 2 escritor iita 25 B Creating a Casework Analysis Method with GeneMapper ID Software 26 C Creating a Databasing or Paternity Analysis Method with Genellappert 1 DoW fELo edid ades etree event ie bibendi eatis 30 D Sample Analysis Using the GeneScan Software and Windows Operating 5y5LeEHS Lessspee iiiter eniin itd bebe drip d 32 E Sample Analysis Using the GeneScan Software and Macintosh Operating DySIeHIS sessiyasini roerei anasa oai 34 F Sample Analysis Using the Genotyper Software an Power Typer Y MOBROL users ca esperto IDE 35 PE CO 38 f 38 Ts Jro bleshoo no rnent n t Parvman Pur Ud 41 A Amplibcatonand Fragment Derechota ina eea toten 41 b GeneMapper JD Amalysis SOW ATE arees roie eimi togam eirmod E Oe abdo UN 47 M Co cnc c S 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 TMD018 Revised 5 12 Page 1 arp Gm
61. ior to multiplex analysis may provide some information as to the ratio of male to female DNA Amelogenin is available as a monoplex amplification with a ladder labeled with either fluorescein Cat DC5171 or TMR Cat DC6171 4 4 Amplification Setup The use of gloves and aerosol resistant pipette tips is highly recommended to prevent cross contamination Keep all pre amplification and post amplification reagents in separate rooms Prepare amplification reactions in a room dedicated for reaction setup Use equipment and supplies dedicated for amplification setup Meticulous care must be taken to ensure successful amplification A guide to amplification troubleshooting is provided in Section 7 A The concentration of 2800M Control DNA was determined by measuring absorbance at 260nm 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 pl or less 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 TMD018 Revised 5 12 Page 7 Promega 4 A Amplification Setup continued 1 Thaw the Gold ST R 10X Buffer and PowerPlex Y 10X Primer Pair Mix Notes 1 Mix reagents by vortexing each tube for 15 seconds before each use Do not centrifuge the 10X Primer P
62. lect PreRun Allow the gel to pre run for 15 20 minutes or until the gel temperature is at least 40 C Open the status window to monitor the gel temperature 8 Prepare samples and allelic ladder samples during the gel pre run Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Revised 5 12 Partt TMD018 Page 23 Promega 5 D Detection of Amplified Fragments Using the ABI PRISM 377 DNA Sequencer continued Sample Preparation and Loading Prepare a loading cocktail by combining and mixing ILS 600 and Blue Dextran Loading Solution as follows 0 5ul ILS 600 x lanes 1 5ul Blue Dextran Loading Solution x s lanes 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 Vortex for 10 15 seconds to mix Combine 2 0ul of prepared loading cocktail and 1 0ul of amplified sample Note Instrument detection limits vary therefore the amount of product mixed with loading cocktail may need to be adjusted If peak heights are higher than desired samples can be diluted in Gold STXR 1X Buffer before mixing with loading cocktail The use of too much template DNA may result in uneven allele peak heights across loci For best results use less template DNA in the amplification reactions or reduce t
63. lic ladders contain fragments of the same lengths as many known alleles for the locus Allelic ladder sizes and repeat units are listed in Table 5 Section 9 A Analysis using GeneScan analysis software and Genotyper software allows allele determination by comparing amplified sample fragments with allelic ladders and internal lane standards When using an internal lane standard the calculated lengths of allelic ladder components may differ from those listed in the table This is due to differences in migration resulting from sequence differences between the allelic ladder fragments and internal size standard and is not a matter of concern Double click on the Allelic Ladders macro A plots window will open to display the blue fluorescein dye allelic ladders i e DYS391 DYS389I DY5439 and DYS380II green JOE dye allelic ladder i e DYS438 DY5437 DYS19 and DYS392 and yellow TMR dye allelic ladders i e DYS393 DYS390 and DYS385 Confirm that the correct allele designations were assigned to the allelic ladders Figure 10 in Section 6 H Note The software uses one ladder sample to determine allele sizes The macro uses the first ladder sample imported for allele designations If the POWER macro is run a second time the software will use the second ladder if the POWER macro is run a third time the software will use the third ladder etc until all ladders in the project are used If an allelic ladder fails to be analyzed or if
64. 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 Ware D et al 1991 Tetranucleotide repeat polymorphism at the human actin related pseudogene 2 actbp2 detected using the polymerase chain reaction Nucl Acids Res 19 6980 5 Ausubel F M et al 1993 Unit 15 The polymerase chain reaction In Current Protocols in Molecular Biology Vol 2 Greene Publishing Associates Inc and John Wiley and Sons NY 6 Sambrook J Fritsch E F and Maniatis T 1989 Chapter 14 In vitro amplification of DNA by the polymerase chain reaction In Molecular Cloning A Laboratory Manual Second Edition Cold Spring Harbor Laboratory Press Cold Spring Harbor New York 7 PCR Technology Principles and Applications for DNA Amplification 1989 Erlich H A ed Stockton Press New York NY 8 PCR Protocols A Guide to Methods and Applications 1990 Innis M A et al eds Academic Press San Diego CA 9 Gusm o L and Carracedo A 2003 Y chromosome specific STRs Profiles in DNA 6 1 3 6 10 Jobling M A Pandya A Tyler Smith C 1997 The Y
65. m Part TMD018 Page 2 Printed in USA Revised 5 12 The PowerPlex Y System provides all of the materials necessary to amplify Y STR C regions of purified human genomic DNA except for AmpliTaq Gold DNA So polymerase This manual contains separate protocols for use of the PowerPlex Y System with the Perkin Elmer model 480 and GeneAmp PCR system 9600 9700 and Promega 2400 thermal cyclers in addition to protocols to separate amplified products and detection of separated material Figure 1 Protocols to operate the fluorescence detection instruments should be obtained from the instrument manufacturer Information on other Promega fluorescent STR systems is available upon request from Promega or online at www promega com Amplification Setup Y Section 4 A Thermal Cycling Section 4 B GeneAmp PCR System 9700 GeneAmp PCR System 9600 GeneAmp PCR System 2400 Model 480 Thermal Cycler Instrument Setup and Sample Preparation Section 5 Applied Biosystems 3130 ABI PRISM 3100 or 3100 ABI PRISM 3100 Genetic or 3130x Genetic Analyzer Avant Genetic Analyzer with Analyzer with Data Collection with Data Collection Data Collection Software Software Version 1 0 1 or 1 1 Software Version 3 0 Version 2 0 Section 5 B Section 5 A Section 5 A ABI PRISM 310 Genetic ABI PRISM 377 DNA Analyzer Sequencer Section 5 C Section 5 D Data Analysis Section 6 GeneMapper D Software GeneScan Software and GeneScan Softw
66. many off ladder alleles are found in the samples samples should be re analyzed using another ladder from the project 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 TMD018 Page 36 Printed in USA Revised 5 12 7 Double click on the Display Fluorescein Data macro to display the blue C dye for all sample injections or lanes Scroll down to observe and edit as ke a Promega 8 Double click on the Display TMR Data macro to display the yellow dye for all sample injections or lanes Scroll down to observe and edit as needed 9 Double click on the Display JOE Data macro to display the green dye for all sample injections or lanes Scroll down to observe and edit as needed 10 Create the appropriate table by selecting the PowerTable Make Allele Table or Make Vertical Table macro The three available table formats are shown below The PowerTable option allows up to four alleles per sample file Additional information such as low peak signal or high peak signal is also included The Allele Table and Vertical Table options include only two alleles per locus If more than two alleles are present at a locus the smallest alleles identified are included The Allele Table format displays the categories loci in columns while the Vertical table format displays the categories in rows These tables can be customized to fit needs To
67. ment sensitivities can vary The injection time and voltage may be adjusted in the Module Manager A suggested range for the injection time is 3 22 seconds and for the injection voltage is 1 3kV 2 In the Protocol Manager select New Type a name for your protocol Select Regular in the Type drop down list and select the run module you created in the previous step in the Run Module drop down list Lastly select F in the Dye Set drop down list Select OK 3 Inthe 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 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 TMD018 Revised 5 12 Page 13 e 5 A Detection of Amplified Fragments Using the ABI PRISM 3100 or 3100 Avant ke Genetic Analyzer with Data Collection Software Version 2 0 or the Applied Pye Biosystems 3130 or 3130x Genetic Analyzer with Data Collection Software m g Version 3 0 continued 4 Inthe GeneMapper plate record enter sample names in the appropriate cells Scroll to the right I
68. monium persulfate 0 1mM EDTA pH 8 0 to 500ul of deionized water 121g Tris base 0 037 EDTA Na EDTA 2H 0 Dissolve Tris base and EDTA in Blue Dextran Loading Solution 88 25 formamide 15mg ml blue dextran 900ml of deionized water Adjust to d d iU pH 8 0 with HCl Bring the final Gold STXR 10X Buffer volume to 1 liter with deionized 500mM KCI water 100mM Tris HCI TE buffer with 20ug ml glycogen pH 8 3 at 25 C g ml glycog 15mM MgCl 1 21g Tris base 1 Triton X 100 0 037g EDTA 2mM each dNTP Na EDTA 2H 0 1 6mg ml BSA 20ug ml glycogen TBE 10X buffer Dissolve Tris base and EDTA in se Dusbasss 900ml of deionized water Adjust to 7 Us EDTA pH 8 0 with HCI Add glycogen Na EDTA 2H O Bring the final volume to 1 liter with 550g boric acid deionized water Dissolve Tris base and EDTA in 800ml of deionized water Slowly add the boric acid and monitor the pH until the desired pH of 8 3 is obtained Bring the final volume to 1 liter with deionized water 9 F Related Products Fluorescent STR Multiplex Systems Product Size Cat PowerPlex 16 System 100 reactions DC6531 400 reactions DC6530 PowerPlex 21 System 200 reactions DC8902 PowerPlex 18D System 200 reactions DC1802 800 reactions DC1808 PowerPlex 16 HS System 100 reactions DC2101 400 reactions DC2100 Not for Medical Diagnostic Use Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526
69. n the Results Group 1 column select the desired results group In the Instrument Protocol 1 column select the protocol you created in Step 2 Be sure this information is present for each row that contains a sample name Select OK Note To create a new Results Group select New in the drop down menu in the Results Group column Select the General tab and enter a name Select the Analysis tab and select GeneMapper Generic in the Analysis type drop down list 5 Place samples in the instrument and close the instrument doors 6 Inthe spectral viewer confirm that dye set F is active and set the correct active calibration for dye set F 7 Inthe 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 windows in the data collection software Each injection will take approximately 45 minutes Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 95
70. o Be Supplied by the User e model 480 or GeneAmp PCR System 9600 9700 or 2400 thermal cycler Promega Applied Biosystems e microcentrifuge 0 5ml or 0 2ml thin walled reaction tubes or MicroAmp optical 96 well reaction plate Applied Biosystems aerosol resistant pipette tips see Section 9 F AmpliTaq Gold DNA polymerase Applied Biosystems Nuclease Free Water Cat P1193 Mineral Oil Cat DY1151 for use with the model 480 thermal cycler We routinely amplify 0 5 1ng of male template DNA in a 25yl reaction volume using the protocols detailed below Expect to see high peak heights at the smaller loci and relatively lower peak heights at larger loci if more than the recommended amount of male template DNA is used Reduce the amount of male template DNA or the number of cycles to correct this The PowerPlex Y System is optimized for the GeneAmp PCR System 9700 thermal cycler Amplification protocols for the GeneAmp PCR Systems 9600 and 2400 thermal cyclers and Perkin Elmer model 480 thermal cycler also are provided A mixture of male and female DNA will often necessitate the use of more than 1ng of total DNA male and female DNA combined This system has been designed to amplify a male derived haplotype even in the presence of female DNA The range of total input DNA and the ratio of male to female DNA that produces acceptable results should be validated in your laboratory Amplification and analysis of the Amelogenin locus pr
71. ogram and analyzed using the PowerTyper Y Macro Release 2 0 The PowerTyper Y Macro Release 2 0 can be downloaded from the Promega web site at www promega com resources tools powerty per macros The PowerTyper Y Macro Release 2 0 is used in conjunction with Macintosh Genotyper software version 2 5 and Windows NT Genotyper software version 3 6 or later The Genotyper software must be installed on your computer before the PowerTyper Y Macro Release 2 0 can be used Be certain the Sample Info Macintosh computers or Color Info Windows NT operating systems column for each lane containing allelic ladder mix contains the word ladder The macro uses the word ladder to identify the sample file s containing allelic ladder Sample info can be added or modified after importing into the PowerTyper Macro Highlight the sample then select show dye lanes window in the Views menu 1 Transfer the PowerTyper Y Macro Release 2 0 to a designated location on your computer hard drive 2 Open the Genotyper software then the PowerTyper Y Macro For questions about the Genotyper software refer to the Genotyper Analysis Software User s Manual 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 TMD018 Revised 5 12 Page 35 Promega 6 F Sample Analysis Using the Geno
72. on 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 The Internal Lane Standard 600 The Internal Lane Standard 600 contains 22 DNA fragments of 60 80 100 120 140 160 180 200 225 250 275 300 325 350 375 400 425 450 475 500 550 and 600 bases in length Figure 13 Each fragment is labeled with carboxy X rhodamine CXR and may be detected separately as a fourth color in the presence of PowerPlex Y amplified material The ILS 600 is designed for use in each gel lane or CE injection to increase precision in analyses when using the PowerPlex Y System Protocols for preparation and use of this internal lane standard are provided in Section 5 Note The PowerPlex Y System requires detection and definition of the 375 base peak of the ILS 600 to accurately size the largest alleles that may be observed 600 60 80 120 140 160 180 229 00 2 5 325 350 375 425 450 475 400 200 5751TA Figure 13 Internal Lane Standard 600 An electropherogram showing the Internal Lane Standard 600 fragments 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 TMD018 Page 52 Pr
73. or voltage in the run module If peak heights are higher than desired samples can be diluted in Gold STXR 1X Buffer before mixing with loading cocktail The use of too much template DNA may result in uneven allele peak heights across loci For best results use less DNA template in the amplification reactions or reduce the number of cycles in the amplification program by 2 4 cycles to achieve the desired signal intensity Centrifuge plate briefly to remove air bubbles from the wells Denature samples at 95 C for 3 minutes then immediately chill on crushed ice or in an ice water bath for 3 minutes Denature samples just prior to loading the instrument Instrument Preparation Refer to the ABI PRISM 3100 Genetic Analyzer User s Manual for instructions on cleaning the blocks installing the capillary array performing a spatial calibration and adding polymer to the reserve syringe 1 2 Open the ABI PRISM 3100 Data Collection Software Change the GeneScan36 POP4DefaultModule module run time to 2 000 seconds Change the injection voltage to 3kV Change the injection time to 11 seconds Note Instrument sensitivities can vary Injection time and voltage may be adjusted in the Module Manager A suggested range for the injection time is 3 22 seconds and for the injection voltage is 1 3kV Save the module with a new name e g GeneScan36 POPA4PowerPlexY 3kV 11secs 2000 Use this as the initial run module for all runs Open
74. ple sheet as described in the ABI PRISM 310 Genetic Analyzer User s Manual Enter the appropriate sample information in the Sample Info column For rows containing PowerPlex Y Allelic Ladder Mix insert the word ladder in the Sample Info column for the blue dye color yellow dye color and green dye color This information must be entered to successfully analyze your data using the PowerTyper Y Macro 3 Create a new GeneScan injection list Select the appropriate sample sheet by using the drop down menu 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 TMD018 Revised 5 12 Page 19 Promega 5 C Detection of Amplified Fragments Using the ABI PRISM 310 Genetic Analyzer continued 4 Select the GS STR POP4 1ml A Module using the drop down menu Change the injection time to the appropriate setting and the run time to 27 minutes Keep the settings for the remaining parameters as shown below Inj Secs 2 5 Inj kV 15 0 Run kV 15 0 Run C 60 Run Time 27 You may need to optimize the injection time for individual instruments Injection times of 2 5 seconds are recommended for samples that contain 0 5 1ng of male template DNA Allelic ladder and samples amplified with less than 32 cycles may work best with longer injection times 5 seconds Use of highly sensitive instrumen
75. poration Products may be covered by pending or issued patents or may have certain limitations Please visit our Web site for more information All prices and specifications are subject to change without prior notice Product claims are subject to change Please contact Promega Technical Services or access the Promega online catalog for the most up to date information on Promega products 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 TMD018 Revised 5 12 Page 57
76. roduct does not convey a license to use AmpliTaq Gold DNA polymerase You should purchase AmpliTaq Gold DNA polymerase licensed for the forensic and human identity field directly from your authorized enzyme supplier 2003 2007 2008 2011 2012 Promega Corporation All Rights Reserved Maxwell Plexor and PowerPlex are registered trademarks of Promega Corporation Differex DNA IO PowerTyper and Slicprep are trademarks of Promega Corporation ABI PRISM GeneMapper GeneScan Genotyper and MicroAmp are registered trademarks of Applera Corporation AmpliTaq Gold and GeneAmp are registered trademarks of Roche Molecular Systems Inc ART is a registered trademark of Molecular Bio Products Inc FTA is a registered trademark of Flinders Technologies Pty Ltd and is licensed to Whatman GenBank is a registered trademark of the U S Dept of Health and Human Services Hi Di and POP 4 are trademarks of Applera Corporation Liqui Nox is a registered trademark of Alconox Inc Long Ranger and Long Ranger Singel are registered trademarks of Cambrex Corporation Macintosh is a registered trademark of Apple Computer Inc Microsoft Windows and Windows NT are registered trademarks of Microsoft Corporation Nalgene is a registered trademark of Nalge Nunc International Standard Reference Material is a registered trademark of National Institute of Standards and Technology Triton is a registered trademark of Union Carbide Chemicals and Plastics Technology Cor
77. rporation 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 TMD018 Revised 5 12 Page 51 Promega 9 B oC 1 200 1 000 800 DNA Extraction and Quantitation Methods and Automation Support The DNA IQ System Cat DC6700 is a DNA isolation system designed specifically for forensic and paternity samples 33 This novel system uses paramagnetic particles to prepare clean samples for STR analysis easily and efficiently and can be used to extract DNA from stains or liquid samples such as blood or solutions The DNA IQ Resin eliminates PCR inhibitors and contaminants frequently encountered in casework samples With larger samples the DNA IQ System delivers a consistent amount of total DNA The system has been used to isolate and quantify DNA from routine sample types including buccal swabs stains on FTA paper and liquid blood Additionally DNA has been isolated from casework samples such as tissue differentially separated sexual assault samples and stains on support materials The DNA IQ System has been tested with the PowerPlex Systems to ensure a streamlined process See Section 9 F for ordering information For applications requiring human specific DNA quantification the Plexor HY System Cat DC1000 has been developed 34 See Section 9 F for ordering information For information about automati
78. rs caused by nontemplated nucleotide addition by Tag DNA polymerase Genome Res 5 312 7 29 Magnuson V L et al 1996 Substrate nucleotide determined non templated addition of adenine by Tag DNA polymerase Implications for PCR based genotyping BioTechniques 21 700 9 30 Walsh P S Fildes N J and Reynolds R 1996 Sequence analysis and characterization of stutter products at the tetranucleotide repeat locus vWA Nucl Acids Res 24 2807 12 31 Moller A Meyer E and Brinkmann B 1994 Different types of structural variation in STRs HumFES FPS HumVWA and HumD21811 Int J Leg Med 106 319 23 32 Brinkmann B Moller A and Wiegand P 1995 Structure of new mutations in 2 STR systems Int J Leg Med 107 201 3 33 Mandrekar P V Krenke B E and Tereba A 2001 DNA IQ The intelligent way to purify DNA Profiles in DNA 4 3 16 34 Krenke B E et al 2005 Development of a novel fluorescent two primer approach to quantitative PCR Profiles in DNA 8 1 3 5 Additional STR references can be found at www promega com geneticidentity Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD018 Revised 5 12 Page 49 Promega Appendix 9 A Advantages of Using the Loci in the PowerPlex Y System The loci included in the PowerPlex Y System Tables 4 and 5
79. s as well The PowerPlex Y System is used for human identification applications including forensic analysis relationship testing and research use This system allows co amplification and three color detection of twelve loci DYS19 DYS385a b DYS389I 1I DYS390 DYS391 DYS392 DYS393 DYS437 DYS438 and DYS439 18 One primer specific for each of the DYS389I TI DYS391 and DYS439 loci is labeled with fluorescein FL one primer specific for each of the DYS385a b DYS390 and DYS393 loci is labeled with carboxy tetramethyl rhodamine TMR and one primer specific for each of the DYS19 DYS392 DYS437 and DYS438 loci is labeled with 6 carboxy 4 5 dichloro 2 7 dimethoxy fluorescein JOE All twelve loci are amplified simultaneously in a single tube and analyzed in a single injection or gel lane The PowerPlex Y System is compatible with the ABI PRISM 310 3100 and 3100 Avant Genetic Analyzers Applied Biosystems 3130 and 3130xI Genetic Analyzers and ABI PRISM 377 DNA Sequencer The protocols presented in this manual were tested at Promega Corporation Amplification and detection instrumentation may vary You may need to optimize protocols including cycle number and injection conditions or loading volume for each laboratory instrument In house validation should be performed 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 co
80. tation amplification of gt 1ng male template or use of 32 cycles may require shorter injection times Note Migration of fragments may vary slightly over the course of a long ABI PRISM 310 Genetic Analyzer run This may be due to changes in temperature or changes in the column When analyzing many samples injections of allelic ladder at different times throughout the run can aid in accurately genotyping the samples Select the appropriate matrix file Section 3 B To analyze data automatically select the auto analyze checkbox and the appropriate analysis parameters and size standard Refer to the ABI PRISM 310 Genetic Analyzer User s Manual for specific information on these options After loading the sample tray and closing the doors select Run to start the capillary electrophoresis system Monitor electrophoresis by observing the raw data and status windows Each sample will take approximately 35 minutes for syringe pumping sample injection and sample electrophoresis Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Partt TMD018 Page 20 Printed in USA Revised 5 12 5 D Detection of Amplified Fragments Using the ABI PRISM 377 DNA C Sequencer e Materials to Be Supplied by the User Promega Solution compositions are provided in Section 9 E Long Ranger gel solution Cambrex Cat 50611 or Long
81. the instrument doors 14 Locate the pending plate record that you just created and click once on the name 15 Once the pending plate record is highlighted click on the plate graphic that corresponds to the plate on the autosampler that contains your amplified samples to link the plate to the plate record 16 When the plate record is linked to the plate the plate graphic will change from yellow to green the plate record moves from the pending plate records table to the linked plate records table and the Run Instrument button becomes enabled 17 Select Run Instrument on the toolbar to start the sample run 18 Monitor electrophoresis by observing the run status array and capillary views windows in the collection software Each injection will take approximately 45 minutes 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 TMD018 Revised 5 12 Page 17 Promega 5 C Detection of Amplified Fragments Using the ABI PRISM 310 Genetic Analyzer Materials to Be Supplied by the User 95 C dry heating block water bath or thermal cycler 310 capillaries 47cm x 50um performance optimized polymer 4 POP 4 10X genetic analyzer buffer with EDTA sample tubes and septa aerosol resistant pipette tips Hi Di formamide Applied Biosystems Cat 4311320 PowerPlex Matrix Standards 310 Cat
82. thod were not in the same Either panel size standard mode Classic vs Basic or Advanced Be sure both files or analysis method is invalid are set to the same mode either Classic or Basic or Advanced 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 Standards 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 Il T T T T pme I export TENET PowerPlex 15 v4 ILE60 adv 8104 ffc ct2j06 em 03 Run Folder ASHO T EE oF Ca m 1 5 F Size Match Editar E 3 eszi Size ms File Edt Yew Tools MT St l l nce LP o AG ar rs REN em pP osgn Size Matohes Size Calling cures asso Sizing Quality 0 0 Deride 0 I Aerio f 276 qme 95 xs 160 eas 300328 ax 5686TA Figure 12 An example showing improper assignment of size standard fragments in the GeneMapper ID software Promega Corpora
83. tion 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Partt TMD018 Revised 5 12 Page 45 7 B GeneMapper ID Analysis Software continued Symptoms Causes and Comments The bins text file assigned to the analysis method may have Error message Both the Bin Set used in the Analysis Method and the Panel must belong to the same Chemistry Kit Significantly raised baseline been deleted In the GeneMapper Manager select the Analysis Methods tab and open the analysis method of interest Select the Alleles tab and select an appropriate bins text file The wrong bins text file was chosen in the analysis method Allele tab Be sure to choose the appropriate bins text file as shown in Figure 3 Poor spectral calibration for the ABI PRISM 3100 and 3100 Avant Genetic Analyzers and Applied Biosystems 3130 and 3130xI Genetic Analyzers Perform a new spectral calibration and re run the samples Poor matrix for the ABI PRISM 310 Genetic Analyzer Re run and optimize the matrix Use of Classic mode analysis method Use of Classic mode analysis on samples can result in baselines with more noise than those analyzed using the Basic or Advanced mode analysis method Advanced mode analysis methods and size standards are recommended If none of the samples had matrices applied when run on the Red bar appears during an
84. to assist movement of solution to the bottom of the plates and to prevent formation of bubbles Insert a 36 well sharkstooth comb or 34 well square tooth comb between the glass plates Sharkstooth combs with 64 or 96 wells also may be used Secure the comb with 3 evenly spaced clamps Keep the remaining acrylamide solution as a polymerization control Allow polymerization to proceed for at least 2 hours Check the polymerization control to be sure that polymerization has occurred Note The gel may be stored overnight if a paper towel saturated with deionized water and plastic wrap are placed around the top and bottom to prevent the gel from drying out crystallization of the urea will destroy the gel Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD018 Page 22 Printed in USA Revised 5 12 Instrument Preparation C 2 1 Open the ABI PRISM 377 Data Collection Software 2 Prepare a sample sheet as described in the GeneScan Analysis Software m g User s Manual Enter the appropriate sample information in the Sample Info column For lanes containing PowerPlex Y Allelic Ladder Mix insert the word ladder in the Sample Info column for the blue dye color yellow dye color and green dye color This information must be entered to successfully analyze your data using the PowerTyper Y Macro Release 2 0
85. trophoresis Promega 2 Monitor electrophoresis by observing the gel image and status windows 3 Allow electrophoresis to proceed for 2 5 hours 4 Track and extract the gel lanes Reuse of Glass Plates Separate the glass plates and discard the gel Clean glass plates with hot water and a detergent such as 1 Liqui Nox detergent Rinse extremely well with deionized water and allow the plates to air dry Do not scrape plates with abrasive materials during this process Note Soap and oil may build up on plates resulting in gel extrusion or hazy background Soak plates in 2N HCI for 15 minutes then rinse thoroughly to remove any buildup 6 Data Analysis 6 A PowerPlex Panels and Bins Text Files with GeneMapper ID Software Version 3 2 To facilitate analysis of data generated with the PowerPlex Y System we have created panels and bins text files to allow automatic assignment of genotypes using GeneMapper ID software version 3 2 We recommend that users of GeneMapper ID software version 3 2 complete the Applied Biosystems GeneMapper ID Software Human Identification Analysis Tutorial to familiarize themselves with proper operation of the software For GeneMapper ID software version 3 1 users we recommend upgrading to version 3 2 Getting Started 1 Obtain the proper panels and bins text files for use with GeneMapper ID from the Promega web site at www promega com resources tools genemapper id software panels an
86. ts of the PowerPlex Y System are shown in Figure 9 The PowerPlex Y Allelic Ladder Mix is shown in Figure 10 A DYS391 DY 3891 DYS439 o DYS3891 a 30 110 130 150 170 190 210 230 250 270 290 310 330 B DYS438 DYS437 DYS19 DYS392 30 110 130 150 170 130 210 230 250 270 290 310 330 4000 3000 000 1000 0 ul C DY 393 DY 390 2 2 6360TA Figure 9 The PowerPlex Y System A single source male template DNA 0 5ng was amplified using the PowerPlex Y System 10X Primer Pair Mix Amplification products were detected using an Applied Biosystems 3130 Genetic Analyzer using a 3kV 3 second injection Results were analyzed using GeneMapper ID software version 3 2 Panel A An electropherogram showing the peaks of the fluorescein labeled loci DYS391 DYS389I DYS439 and DYS389II Panel B An electropherogram showing the peaks of the JOE labeled loci DYS438 DYS437 DYS19 and DYS392 Panel C An electropherogram showing the peaks of the TMR labeled loci DYS393 DYS390 and DYS385 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 TMD018 Printed in USA Page 38 Revised 5 12 30 110 130 130 170 1830 z10 230 230 270 290 210 320 330 i Promega 600 400 200 0 f A E el leijui 13 C e Jer r2 14 24 pe ps Bal Ball Ba 10 12 vat 30 110 130 13
87. typer Software and PowerTyper Y Macro continued 3 In the File menu select Import and import the GeneScan project or sample files to be analyzed Import the blue yellow green and red dye colors Note To select the dye colors to be imported select Set Preferences in the Edit menu Double click on the Check ILS macro The macros are listed at the bottom left corner of the active window A plots window will be displayed to show the internal lane standard i e ILS 600 in the red dye color Scroll down to view and confirm that the internal lane standard fragment sizes are correct If necessary re analyze samples using the GeneScan software and redefine internal lane standard fragments Note The software uses one ladder sample to determine allele sizes The macro uses the first ladder sample imported for allele designations For casework double click on the POWER macro The POWER macro identifies alleles in the ladder sample and calculates offsets for all loci This process may take several minutes When completed a plots window will open to display the allelic ladders i e DYS391 DYS389I DYS439 DYS389II etc Alternatively for databasing or paternity double click on the POWER 20 Filter macro This macro has a higher level of filtering than the standard POWER macro to reduce the need for manual editing of peak labels The POWER 20 Filter should not be used if mixtures may exist In general alle
88. u ef epe e 7 5726TA Figure 6 The Size Standard Editor 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 TMD018 Revised 5 12 Page 29 C 6 B Creating a Casework Analysis Method with GeneMapper ID Software e Version 3 2 continued Promega k Processing Sample Data for Casework L Import sample files into a new project as described in the Applied Biosystems GeneMapper ID Software Human Identification Analysis Tutorial In the Sample Type column use the drop down menu to select Ladder Sample Positive Control or Negative Control Every folder in the project must contain at least one allelic ladder that is designated as such for proper genotyping In the Analysis Method column select the analysis method created previously in the Creating a Casework Analysis Method section In the Panel column select PowerPlex_Y_ID3 2 X where X refers to the most recent version of the panels files This is the panels text file that was imported in Section 6 A In the Size Standard column select the size standard that was created in Creating a Size Standard section If analyzing data from an ABI PRISM 310 Genetic Analyzer or an ABI PRISM 377 DNA Sequencer ensure that the appropriate matrix file is selected in the Matrix column Select Analyze
89. y glasses when working with formamide Sample Preparation 1 Prepare a loading cocktail by combining and mixing Internal Lane Standard 600 and Hi Di formamide as follows 0 5ul ILS 600 x injections 9 5ul Hi Di formamide x injections Note The volume of internal lane standard used in the loading cocktail can be increased or decreased to adjust the intensity of the size standard peaks The optimal peak height for the 100 base fragment of the internal lane standard is 500 1 000RFU If peak heights are too low we recommend altering the formamide internal lane standard mix to contain 1 0ul of ILS 600 and 9 0ul of Hi Di formamide If peak heights are too high we recommend altering the loading cocktail to contain 0 25u1 of ILS 600 and 9 75ul of formamide 2 Vortex for 10 15 seconds to mix 3 Pipet 10ul of formamide internal lane standard mix into each well Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD018 Printed in USA Page 12 Revised 5 12 4 Add 1pl of amplified sample or 1ul of PowerPlex Y Allelic Ladder Mix C Cover wells with appropriate septa ke Note Instrument detection limits vary therefore injection time injection Promega voltage or the amount of product mixed with loading cocktail may need to be adjusted Use the Module Manager in the data collection software to

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