Home

PowerPlex® Y System

1. 11 A Detection of Amplified Fragments Using the ABI PRISM 3100 or 3100 Avant Genetic Analyzer with Data Collection Software Version 2 0 and the Applied Biosystems 3130 or 3130xl Genetic Analyzer with Data Collection Software Version 3 0 11 B Detection of Amplified Fragments Using the ABI PRISM 3100 Genetic Analyzer with Data Collection Software Version 1 0 1 or 1 1 14 C Detection of Amplified Fragments Using the ABI PRISM 310 Genetic Analyzer 16 D Detection of Amplified Fragments Using the ABI PRISM 377 DNA Sequencer 19 VI Data Analysis 23 A PowerPlex Panel and Bin Sets with GeneMapper ID Software Version 3 2 23 B Creating a Casework Analysis Method with GeneMapper ID Software 24 C Creating a Databasing or Paternity Analysis Method with GeneMapper ID Software 28 D Sample Analysis Using the Gene
2. 5 Navigate to the saved panel and bin files Select Promega_Panels_ID3 2 X txt where X refers to the most recent version of the panel and bin files Select Import 6 In the navigation pane highlight the Promega_Panels_ID3 2 X folder that you just imported 7 Select File then Import Bin Set 8 Navigate to the saved panel and bin files Select Promega_Bins_ID3 2 X txt then Import 9 At the bottom of the Panel Manager window select Apply then OK The panel manager window will close automatically VI B Creating a Casework Analysis Method with GeneMapper ID Software For detailed instructions see the Applied Biosystems GeneMapper ID software tutorial 1 Select Tools then GeneMapper Manager 2 Select the Analysis Methods tab 3 Select New and a new analysis method dialog box will open 4 Select HID and select OK Note If you do not see the HID option you do not have the GeneMapper ID software Contact Applied Biosystems 5 Enter a descriptive name for the analysis method such as PowerPlexY advanced 6 Select the Allele tab Figure 3 7 Select the bin set corresponding to the PowerPlex System Promega_Bins_ID3 2 X where X refers to the most recent version of the bin set 8 Ensure that the Use marker specific stutter ratio if available box is checked Page 24 t
3. 2 Select the Size Standard tab 3 Select New 4 Select Basic or Advanced Figure 5 The type of analysis method selected must match the type of analysis method created earlier Select OK 5 Enter a detailed name such as ILS 600 advanced in the Size Standard Editor Figure 6 6 Choose red as the color for the size standard dye 7 Enter the sizes of the internal lane standard fragments see Section IX D Figure 13 8 Select OK 6362TA Figure 7 The Allele tab with settings for using a 20 peak filter Select the bin set Promega_Bins_ID3 2 X txt where X refers to the most recent version of the bin set tmd018 0508 qxp 8 1 2008 3 25 PM Page 29 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 Revised 5 08 VI C Creating a Databasing or Paternity Analysis Method with GeneMapper ID Software continued 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 In the Sample Type column use the drop down menu to select Ladder Sample Positive Control or Negative Control Every folder in the project must contain at least one ladder that is desig
4. 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 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 STRs Int J Legal Med 114 305 9 Page 46 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 Revised 5 08 tmd018 0508 qxp 8 1 2008 3 25 PM Page 46 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 08 Page 47 12 Roewer L et al 2001 Online reference database of European Y chromosomal short tandem repeat STR haplotypes Forensic Sci Int 118 106 13 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 14 Kayser M et al 1997 Evaluation of Y chromosomal STRs A multicenter study Int J Legal Med 110 125
5. Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD018 Printed in USA Revised 5 08 VI B Creating a Casework Analysis Method with GeneMapper ID Software continued Processing Sample Data for Casework 1 Import sample files into a new project as described in the Applied Biosystems GeneMapper ID Software Human Identification Analysis Tutorial 2 In the Sample Type column use the drop down menu to select Ladder Sample Positive Control or Negative Control Every folder in the project must contain at least one allelic ladder that is designated as such for proper genotyping 3 In the Analysis Method column select the analysis method created previously in the Creating a Casework Analysis Method section 4 In the Panel column select PowerPlex_Y_ID3 2 X where X refers to the most recent version of the panel files This is the panel set that was imported in Section VI A 5 In the Size Standard column select the size standard that was created in 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 data analysis VI C Creating a Databasing or Paterni
6. 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 3 Create a new 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 pull down menus 5 Select the appropriate gel matrix file Section III 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 b
7. 1ng for each sample into the respective tube containing PCR master mix 7 For the positive amplification control dilute 9948 Male DNA to 0 5ng in the desired template DNA volume Pipet 0 5ng of the diluted DNA into a reaction tube containing PCR master mix 8 For the negative amplification control pipet nuclease free water instead of template DNA into a reaction tube containing PCR master mix 9 Optional The 9947A female DNA can be used as a negative control to document male specificity Pipet the desired quantity of DNA dilution may be necessary into an amplification tube containing PCR master mix 10 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 IV B Amplification Thermal Cycling This manual contains protocols for use of the PowerPlex Y System with the 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
8. 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 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 Budowle B et al 1991 Analysis of the VNTR locus D1S80 by the PCR followed by high resolution PAGE Am J Hum Genet 48 137 44 24 Nakamura Y et al 1987 Variable number of tandem repeat VNTR markers for human g
9. DYS389II FL 256 296 24 34 31 DYS393 TMR 104 136 8 16 13 DYS390 TMR 191 227 18 27 24 DYS385 TMR 243 315 7 25 11 14 DYS438 JOE 101 121 8 12 11 DYS437 JOE 183 199 13 17 15 DYS19 JOE 232 268 10 19 14 DYS392 JOE 294 327 7 18 13 1When 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 2Information about the use of 9947A and 9948 DNA as standard DNA templates can be found in reference 29 3Follows the original nomenclature described by Ayub et al 26 tmd018 0508 qxp 8 1 2008 3 25 PM Page 50 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD018 Revised 5 08 Page 51 IX C DNA Extraction and Quantitation Methods The DNA IQ System Cat DC6700 is a DNA isolation and quantitation system designed specifically for forensic and paternity samples 37 This novel system uses paramagnetic particles to prepare clean samples for STR analysis easily and efficiently and can be used to extract DNA from stains or liquid samples such as blood or solutions The DNA IQ
10. POWER 20 Filter should not be used if mixtures may exist In general allelic ladders contain fragments of the same lengths as many known alleles for the locus Allelic ladder sizes and repeat units are listed in Table 5 Section IX B Analysis using GeneScan analysis software and Genotyper software allows allele determination by comparing amplified sample fragments with allelic ladders and internal lane standards When using an internal lane standard the calculated lengths of allelic ladder components 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 6 Double click on the Allelic Ladders macro A plots window will open to display the blue fluorescein dye allelic ladders i e DYS391 DYS389I DYS439 and DYS389II green JOE dye allelic ladder i e DYS438 DYS437 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 VI 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 unti
11. optimize protocols including cycle number and injection time or loading volume for each laboratory instrument Testing at Promega Corporation shows that 10 22 cycles work well for 0 5 1ng of purified DNA templates For higher amounts of input DNA i e FTA paper or to decrease sensitivity fewer cycles such as 10 16 10 18 or 10 20 should be evaluated In house validation should be performed 1 Place the tubes or MicroAmp plate in 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 samples at 20 C in a light protected box Note Storage of amplified samples at 4 C or higher may produce degradation products tmd018 0508 qxp 8 1 2008 3 24 PM Page 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 Part TMD018 Printed in USA Revised 5 08 Page 10 Protocol for the GeneAmp PCR System 9700 Thermal Cycler1 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 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
12. 14 DYS390 TMR Yq AC011289 TCTG TCTA Complex 14 DYS385a b TMR Yq Z93950 GAAA 14 DYS438 JOE Yq AC002531 TTTTC 26 DYS437 JOE Yq AC002992 TCTA TCTG Complex 26 DYS19 JOE Yq X77751 TAGA Complex 14 DYS392 JOE Yq G09867 TAT 14 1The August 1997 report 27 28 of the DNA Commission of the International Society for Forensic Haemogenetics ISFH states 1 for STR loci within coding genes the coding strand shall be used and the repeat sequence motif defined using the first possible 5 nucleotide of a repeat motif and 2 for STR loci not associated with a coding gene the first database entry or original literature description shall be used TMR carboxy tetramethylrhodamine FL fluorescein JOE 6 carboxy 4 5 dichloro 2 7 dimethoxyfluorescein tmd018 0508 qxp 8 1 2008 3 25 PM Page 49 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 Revised 5 08 Page 50 IX B Advantages of Using the Loci in the PowerPlex Y System continued We have carefully selected primers to avoid or minimize artifacts including those associated with Taq DNA polymerase such as repeat slippage and terminal nucleotide addition Repeat slippage 30 31 sometimes called n 4 bands stutter or shadow bands is due to the loss of a rep
13. 5 Centrifuge plate briefly to remove air bubbles from the wells if necessary 6 Denature samples at 95 C for 3 minutes then immediately chill on crushed ice or in an ice water bath for 3 minutes Denature samples just prior to loading the instrument Instrument Preparation Refer to the ABI PRISM 3100 Genetic Analyzer User s Manual for instructions on cleaning the blocks installing the capillary array performing a spatial calibration and adding polymer to the reserve syringe 1 Open the ABI PRISM 3100 data collection software 2 Change the GeneScan36_POP4DefaultModule module run time to 2 000 seconds 3 Change the injection voltage to 3kV 4 Change the injection time to 11 seconds Note Instrument sensitivities can vary Injection time and voltage may be adjusted in the Module Manager A suggested range for the injection time is 3 22 seconds and for the injection voltage is 1 3kV 5 Save the module with a new name e g GeneScan36_POP4PowerPlexY_3kV_11secs_2000 Use this as the initial run module for all runs 6 Open a new plate record Name the plate and select GeneScan Select the plate size 96 well Select Finish 7 Complete the plate record spreadsheet for the wells you have loaded Enter appropriate information into the sample name and color info columns For allelic ladder samples insert the word ladder into the color info column for the blue yellow and green dy
14. 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 tmd018 0508 qxp 8 1 2008 3 25 PM Page 45 VII 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 la
15. 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 VI B or VI C Panel file selected for analysis was incorrect for the STR system used Assign correct panel file that corresponds to the STR system used for amplification The allelic ladder was not identified as an allelic ladder in the sample type column The wrong analysis type was chosen for the analysis method Be sure to use the HID analysis type The internal lane standard was not properly identified in the sample Manually redefine the sizes of the size standard fragments in the sample Page 42 5685TA Figure 11 The error message that appears in the GeneMapper ID software when the analysis parameters and size standard have different analysis types tmd018 0508 qxp 8 1 2008 3 25 PM Page 42 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD018 Revised 5 08 Page 43 Symptoms Causes and Comments Size standard not called Starting data point was incorrect for the partial range chosen correctly Figure 12 in Section VI B Adjust the starting data point in the analysis method Alternatively use a full range for the analysis Extra peaks in advanced mode size standard
16. Manager then selecting the General tab The analysis type cannot be changed If the method is not HID it should be deleted and a new analysis method created Page 44 tmd018 0508 qxp 8 1 2008 3 25 PM Page 44 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD018 Revised 5 08 Page 45 VII C PowerTyper Y Macro Symptoms Causes and Comments File does not open Genotyper software was not installed Be certain that the on your computer Genotyper software version 2 5 Macintosh or version 3 6 or higher Windows NT is installed Incorrect version of Genotyper software The PowerTyper Y Macro will not work with Genotyper software versions prior to version 2 5 The CD ROM may have been damaged during shipment Contact Technical Services by e mail genetic promega com The file was corrupted during download or transfer Download the file again or obtain the file on CD ROM Error message Allelic ladder sample files were not identified Be certain the Could not complete the sample info or color info column for each lane containing Run Macro command because PowerPlex 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 i
17. Open the size match editor Highlight the extra peak select Edit and select delete size label Select auto adjust sizes Run was too short and larger peaks in ILS were not captured Not all ILS 600 peaks defined in the size standard were detected during the run Create a new size standard using the internal lane standard fragments present in the sample Re run samples using a longer run time Peaks in size standard missing If peaks are below threshold decrease the peak threshold in the analysis method for the red channel to include peaks If peaks are low quality redefine the size standard for the sample to skip these peaks Error message The size standard and analysis method were not in the same Either panel size standard mode Classic vs Basic or Advanced Be sure both files or analysis method is invalid are set to the same mode either Classic or Basic or Advanced mode No alleles called but no error Panel was not selected for sample In the Panel column select message appears the appropriate panel set for the STR system that was used No size standard was selected In the size standards column be sure to select the appropriate size standard Size 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 wi
18. Printed in USA Revised 5 08 II 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 25 l each Includes Pre amplification Components Box Blue Label 1 300 l Gold ST R 10X Buffer 1 125 l PowerPlex Y 10X Primer Pair Mix 25 l 9948 Male DNA 10ng l 25 l 9947A DNA 10ng l Postamplification Components Box Beige Label 1 12 5 l PowerPlex Y Allelic Ladder Mix 1 150 l Internal Lane Standard ILS 600 1 Protocol Product Size Cat PowerPlex Y System 200 reactions DC6760 Not For Medical Diagnostic Use Cat DC6760 contains sufficient reagents for 200 reactions of 25 l each Includes Pre amplification Components Box Blue Label 2 300 l Gold ST R 10X Buffer 4 125 l PowerPlex Y 10X Primer Pair Mix 25 l 9948 Male DNA 10ng l 25 l 9947A DNA 10ng l Postamplification Components Box Beige Label 4 12 5 l PowerPlex Y Allelic Ladder Mix 2 150 l Internal Lane Standard ILS 600 1 Protocol The PowerPlex Y Allelic Ladder Mix is provided in a separate sealed bag for shipping This component should be moved to the postamplification box after opening Storage Conditions Store all components at 20 C in a nonfrost free freezer The PowerPlex Y 10X Primer Pair Mix PowerPlex Y Allelic La
19. 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 IX G for ordering information For applications requiring human specific DNA quantification the Plexor HY System Cat DC1000 has been developed 38 See Section IX G for ordering information The DNA IQ System has been fully automated on the Beckman Coulter Biomek 2000 Laboratory Automation Workstation 39 Biomek 3000 Laboratory Automation Workstation 40 and Tecan Freedom EVO Liquid Handler 41 In addition the DNA IQ Reference Sample Kit for Maxwell 16 Cat AS1040 and DNA IQ Casework Sample Kit for Maxwell 16 are available 42 43 For information on automation of laboratory processes on automated workstations contact your local Promega Branch Office or Distributor contact information available at www promega com worldwide or e mail genetic promega com IX D The Internal Lane Standard 600 The Internal Lane Standard ILS 600 con
20. Version 1 0 1 or 1 1 Materials to Be Supplied by the User 95 C dry heating block water bath or thermal cycler crushed ice or ice water bath aerosol resistant pipette tips 3100 capillary array 36cm performance optimized polymer 4 POP 4 for the 3100 10X genetic analyzer buffer with EDTA MicroAmp optical 96 well plate and septa for the 3100 Hi Di formamide Applied Biosystems Cat 4311320 PowerPlex Matrix Standards 3100 3130 Cat DG4650 The quality of formamide is critical Use Hi Di formamide with a conductivity less than 100 S cm Freeze formamide in aliquots at 20 C Multiple freeze thaw cycles or long term storage at 4 C may cause a breakdown of the formamide Formamide with a conductivity greater than 100 S cm may contain ions that compete with DNA during injection which results in lower peak heights and reduced sensitivity A longer injection time may not increase the signal Caution Formamide is an irritant and a teratogen avoid inhalation and contact with skin Read the warning label and take appropriate precautions when handling this substance Always wear gloves and safety glasses when working with formamide Sample Preparation 1 Prepare a loading cocktail by combining and mixing Internal Lane Standard 600 and Hi Di formamide as follows 0 5 l ILS 600 injections 9 5 l Hi Di formamide injections Note The volume of int
21. have not tested other reaction tubes plates or thermal cyclers Calibrate the thermal cycler heating block if necessary Primer concentration was too low Use the recommended primer concentration Mix the 10X PowerPlex Y Primer Pair for 15 seconds using a vortex mixer before use Poor capillary electrophoresis injection ILS 600 peaks also affected Re inject the sample Check the syringe for leakage Check the laser power Poor quality formamide was used Use only Hi Di formamide when analyzing samples 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 08 Page 39 tmd018 0508 qxp 8 1 2008 3 25 PM Page 39 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 Revised 5 08 VII A Amplification and Fragment Detection continued Symptoms Causes and Comments Extra peaks visible in one Contamination with another template DNA or previously or all color channels amplified DNA Cross contamination can be a problem Use aerosol resistant pipette tips and change gloves regularly Samples were not completely denatured Heat denature samples for the recommended time and cool on crushed ice or in an ice water bath immediately prior to l
22. in Figure 13 in Section IX D Store the size standard in the Size Standards folder 7 Apply the size standard file to the samples then analyze the sample files See Section VI 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 Page 32 Analysis Range Start Defined in Step 2 Stop 10 000 Data Processing Baseline Checked Multicomponent Checked Smooth Options Light1 Peak Detection Peak Amplitude Thresholds2 B Y G R Min Peak Half Width 2pts Size Call Range Min 60 Max 600 Size Calling Method Local Southern Method Split Peak Correction None 1Smooth options should be determined by individual laboratories 2The peak amplitude thresholds are the minimum peak heights that the software will call as a peak Values for the peak amplitude thresholds are usually 50 200RFU and should be determined by individual laboratories tmd018 0508 qxp 8 1 2008 3 25 PM Page 32 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 08 Page 33 Notes 1 Peak heights outside the linear range of the instrument may generate artifact peaks due to instrument saturation i e overloa
23. of ILS 600 and 24 5 l of Hi Di formamide 2 Mix for 10 15 seconds using a vortex mixer 3 Combine 25 0 l of prepared loading cocktail and 1 0 l of amplified sample Note Instrument detection limits vary therefore injection time or the amount of product mixed with loading cocktail may need to be increased or decreased If peak heights are higher than desired samples can be diluted in Gold ST R 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 4 Combine 25 0 l of prepared loading cocktail and 1 0 l of PowerPlex Y Allelic Ladder Mix 5 Centrifuge tubes briefly to remove air bubbles from the wells if necessary 6 Denature samples and ladder by heating at 95 C for 3 minutes and immediately chill on crushed ice or in an ice water bath for 3 minutes Denature samples just prior to loading 7 Assemble the tubes in the appropriate autosampler tray 48 or 96 tube 8 Place the autosampler tray in the instrument and close the instrument doors Instrument Preparation Refer to the instrument users manual for instructions on cleaning the pump block installing the capillary calibrating the autosampler and adding polymer to the syringe 1 Open
24. 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 3130xl 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 time or loading volume for each laboratory instrument In house validation should be performed Page 2 tmd018 0508 qxp 8 1 2008 3 23 PM Page 2 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD018 Revised 5 08 Page 3 The PowerPlex Y System provides all of the materials necessary for amplification of Y STR regions of purified genomic DNA except for AmpliTaq Gold DNA 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 2400 thermal cyclers in addition to protocols for separation of amplified products and detect
25. 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 PowerPlex Y System tmd018 0508 qxp 8 1 2008 3 23 PM Page 1 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 Revised 5 08 IX Appendix 48 A Advantages of STR Typing 48 B Advantages of Using the Loci in the PowerPlex Y System 49 C DNA Extraction and Quantitation Methods 51 D The Internal Lane Standard 600 51 E Preparing the PowerPlex Y System Master Mix 52 F Composition of Buffers and Solutions 53 G Related Products 53 I Description STR short tandem repe
26. 0 base fragment of the internal lane standard is 500 1 000RFU If peak heights are too low we recommend altering the formamide internal lane standard mix to contain 1 0 l of ILS 600 and 9 0 l of Hi Di formamide If peak heights are too high we recommend altering the loading cocktail to contain 0 25 l of ILS 600 and 9 75 l of formamide 2 Mix for 10 15 seconds using a vortex mixer 3 Pipet 10 l of formamide internal lane standard mix into each well tmd018 0508 qxp 8 1 2008 3 24 PM Page 11 V A Detection of Amplified Fragments Using the ABI PRISM 3100 or 3100 Avant Genetic Analyzer with Data Collection Software Version 2 0 and the Applied Biosystems 3130 or 3130xl Genetic Analyzer with Data Collection Software Version 3 0 continued 4 Add 1 l of amplified sample or 1 l of allelic ladder mix Cover wells with appropriate septa Note Instrument detection limits vary therefore injection time or the amount of product mixed with loading cocktail may need to be increased or decreased Use the Module Manager in the data collection software to modify the injection time or voltage in the run module If peak heights are higher than desired samples can be diluted in Gold ST R 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 c
27. 100 reactions DC6731 400 reactions DC6730 GenePrint Sex Identification System Amelogenin Fluorescein 100 reactions DC5171 GenePrint Sex Identification System Amelogenin TMR 100 reactions DC6171 Not for Medical Diagnostic Use 10 ammonium persulfate Add 0 05g of ammonium persulfate to 500 l of deionized water Blue Dextran Loading Solution 88 25 formamide 15mg ml blue dextran 4 1mM EDTA pH 8 0 Gold ST R 10X Buffer 500mM KCl 100mM Tris HCl pH 8 3 at 25 C 15mM MgCl2 1 Triton X 100 2mM each dNTP 1 6mg ml BSA TBE 10X buffer 107 8g Tris base 7 44g EDTA Na2EDTA 2H2O 55 0g boric acid 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 TE 4 buffer 10mM Tris HCl 0 1mM EDTA pH 8 0 2 21g Tris base 0 037g EDTA Na2EDTA 2H2O Dissolve Tris base and EDTA in 900ml of deionized water Adjust to pH 8 0 with HCl Bring the final volume to 1 liter with deionized water tmd018 0508 qxp 8 1 2008 3 25 PM Page 53 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 Revised 5 08 IX G Related Products continued Accessory Components Product Size Cat PowerP
28. 18 Printed in USA Revised 5 08 VI F Sample Analysis Using the Genotyper 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 4 Double click on the Check ILS macro The macros are listed at the bottom left corner of the active window A plots window will be displayed to show the internal lane standard i e ILS 600 in the red dye color Scroll down to view and confirm that the internal lane standard fragment sizes are correct If necessary re analyze samples using the GeneScan software and redefine internal lane standard fragments Note The software uses one ladder sample to determine allele sizes The macro uses the first ladder sample imported for allele designations 5 For casework double click on the POWER macro The POWER macro identifies alleles in the ladder sample and calculates offsets for all loci This process may take several minutes When completed a plots window will open to display the allelic ladders i e 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
29. 277 2516 www promega com Printed in USA Part TMD018 Revised 5 08 Page 55 ART Aerosol Resistant Tips Product Volume Size tips pack Cat ART 10 Ultramicro Pipet Tip 0 5 10 l 960 DY1051 ART 20E Ultramicro Pipet Tip 0 5 10 l 960 DY1061 ART 20P Pipet Tip 20 l 960 DY1071 ART GEL Gel Loading Pipet Tip 100 l 960 DY1081 ART 100 Pipet Tip 100 l 960 DY1101 ART 100E Pipet Tip 100 l 960 DY1111 ART 200 Pipet Tip 200 l 960 DY1121 ART 1000E Pipet Tip 1 000 l 800 DY1131 a STR loci are the subject of U S Pat No RE 37 984 German Pat No DE 38 34 636 C2 and other patents issued to the Max Planck Gesellschaft zur F rderung der Wissenschaften e V Germany The development and use of STR loci are covered by U S Pat No 5 364 759 Australian Pat No 670231 and other pending patents assigned to Baylor College of Medicine Houston Texas Patents for the foundational PCR process European Pat Nos 201 184 and 200 362 expired on March 28 2006 In the U S the patents covering the foundational PCR process expired on March 29 2005 b The purchase of this product 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 2000 2008 Promega Corporation All Rights Reserved GenePrint Maxwell Plexor and PowerP
30. 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 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 then 60 C for 30 minutes 4 C soak Protocol for the GeneAmp PCR System 9600 Thermal Cycler Protocol for the Perkin Elmer Model 480 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 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 1When using the GeneAmp PCR System 9700 thermal cycler the ramp rates indicated in the cycling program must be set and the program must be run in 9600 ramp mode The ramp rates are set in the Ramp Rate Modification screen W
31. 5 l PowerPlex Y 10X Primer Pair Mix 2 5 l AmpliTaq Gold DNA polymerase2 0 55 l 2 75u template DNA up to 1ng 3 up to 19 45 l total reaction volume 25 l 1Add nuclease free water to the PCR master mix first then add Gold ST R 10X Buffer PowerPlex Y 10X Primer Pair Mix and AmpliTaq Gold DNA polymerase The template DNA will be added at Step 6 2Assumes the AmpliTaq Gold DNA polymerase is at 5u l If the enzyme concentration is different the volume of enzyme must be adjusted accordingly 3Store DNA templates in nuclease free water or TE 4 buffer 10mM Tris HCl pH 8 0 0 1mM EDTA If the DNA template is stored in TE buffer that is not pH 8 0 or contains a higher EDTA concentration the volume of DNA added should not exceed 20 of the final reaction volume Amplification efficiency and quality can be 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 tmd018 0508 qxp 8 1 2008 3 24 PM Page 8 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD018 Revised 5 08 Page 9 5 Pipet PCR master mix into each reaction tube 6 Pipet the template DNA 0 5
32. E mail genetic promega com VII A Amplification and Fragment Detection Symptoms Causes and Comments Faint or absent allele peaks Impure template DNA Because of the small amount of template used this is rarely a problem Depending on the DNA extraction procedure used and sample source inhibitors 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 postPCR cleanup or desalting lower conductivity formamide or reduced amounts of ILS 600 In house validation should be performed for any of these methods Insufficient enzyme activity Use the recommended amount of AmpliTaq Gold DNA polymerase Check the expiration date on the tube label Incorrect amplification program Confirm the amplification 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 Mg2 or EDTA from the DNA sample can negatively affect PCR A change in pH may also affect PCR Store DNA in TE 4 buffer 10mM Tris HCl pH 8 0 0 1mM EDTA or nuclease free water Thermal cycler plate or tube problems Review the thermal cycling protocols in Section IV B We
33. New in the drop down menu in the results group column Select the General tab and enter a name Select the Analysis tab and select GeneMapper Generic in the Analysis type drop down list 5 Place samples in the instrument and close the instrument doors 6 In the spectral viewer confirm that dye set F is active and set the correct active calibration for dye set F 7 In the run scheduler locate the plate record that you just created in Steps 3 and 4 and click once on the name to highlight it 8 Once the plate record is highlighted click the plate 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 tmd018 0508 qxp 8 1 2008 3 24 PM Page 13 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 Revised 5 08 V B Detection of Amplified Fragments Using the ABI PRISM 3100 Genetic Analyzer with Data Collection Software
34. Place samples in the instrument and close the instrument doors 14 Locate the pending plate record that you just created and click once on the name 15 Once the pending plate record is highlighted click on the plate graphic that corresponds to the plate on the autosampler that contains your amplified samples to link the plate to the plate record 16 When the plate record is linked to the plate the plate graphic will change from yellow to green the plate record moves from the pending plate records table to the linked plate records table and the Run Instrument button becomes enabled 17 Select Run Instrument on 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 V C Detection of Amplified Fragments Using the ABI PRISM 310 Genetic Analyzer Materials to Be Supplied by the User 95 C dry heating block water bath or thermal cycler 310 capillaries 47cm 50 m performance optimized polymer 4 POP 4 10X genetic analyzer buffer with EDTA sample tubes and septa aerosol resistant pipette tips Hi Di formamide Applied Biosystems Cat 4311320 PowerPlex Matrix Standards 310 Cat DG4640 crushed ice or ice water bath The quality of formamide is critical Use Hi Di formamide with a conductivity less than 100
35. S cm Freeze formamide in aliquots at 20 C Multiple freeze thaw cycles or long term storage at 4 C may cause a breakdown of the formamide Formamide with a conductivity greater than 100 S cm may contain ions that compete with DNA during injection This results in lower peak heights and reduced sensitivity A longer injection time may not increase the signal Caution Formamide is an irritant and a teratogen avoid inhalation and contact with skin Read the warning label and take appropriate precautions when handling this substance Always wear gloves and safety glasses when working with formamide Page 16 tmd018 0508 qxp 8 1 2008 3 24 PM Page 16 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD018 Revised 5 08 Page 17 Sample Preparation 1 Prepare a loading cocktail by combining Internal Lane Standard 600 ILS 600 and Hi Di formamide as follows 1 0 l ILS 600 injections 24 0 l Hi Di formamide injections Note The volume of internal lane standard used in the loading cocktail can be increased or decreased to adjust the intensity of the size standard peaks The optimal peak height for the 100 base fragment of the internal lane standard is 500 1 000RFU If peak heights are too high we recommend altering the loading cocktail to contain 0 5 l
36. Scan Software and PC Operating Systems 30 E Sample Analysis Using the GeneScan Software and Macintosh Operating Systems 32 F Sample Analysis Using the Genotyper Software and PowerTyper Y Macro 33 G Controls 36 H Results 36 VII Troubleshooting 39 A Amplification and Fragment Detection 39 B GeneMapper ID Analysis Software 42 C PowerTyper Y Macro 45 VIII References 46 All technical literature is available on the Internet at www promega com tbs Please visit
37. T e c h n i c a l M a n u a l PowerPlex Y System INSTRUCTIONS FOR USE OF PRODUCTS DC6760 AND DC6761 PRINTED IN USA Revised 5 08 Part TMD018 tmd018 0508 qxp 8 1 2008 3 23 PM Page 1 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 08 Page 1 I Description 2 II Product Components and Storage Conditions 4 III Before You Begin 5 A Precautions 5 B Matrix Standardization or Spectral Calibration 6 IV Protocols for DNA Amplification Using the PowerPlex Y System 7 A Amplification Setup 7 B Amplification Thermal Cycling 9 V Instrument Setup and Sample Preparation
38. Using the ABI PRISM 377 DNA Sequencer Materials to Be Supplied by the User Solution compositions are provided in Section IX F Long Ranger gel solution Cambrex Cat 50611 or Long Ranger Singel pack for ABI 377 36cm Cambrex Cat 50691 10 Ammonium Persulfate Cat V3131 TEMED TBE 10X buffer Nalgene tissue culture filter 0 2 micron 36cm front and rear glass plates 36cm gel spacers 0 2mm thick 36 well sharkstooth comb or 34 well square tooth comb 0 2mm thick clamps e g large office binder clamps gel loading pipette tips aerosol resistant pipette tips Section IX G Liqui Nox or other detergent PowerPlex Matrix Standards 310 Cat DG4640 Blue Dextran Loading Solution Cat DV4351 crushed ice or ice water bath 95 C dry heating block water bath or thermal cycler Caution 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 c
39. alysis of STR products are potentially hazardous and should be handled accordingly Table 1 describes the potential hazards associated with such reagents tmd018 0508 qxp 8 1 2008 3 24 PM Page 5 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD018 Printed in USA Revised 5 08 III B 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 Applied Biosystems 3130 and 3130xl Genetic Analyzers A matrix must be generated for each individual instrument The PowerPlex Matrix Standards 310 Cat DG4640 is required for matrix standardization for the ABI PRISM 310 Genetic Analyzer and ABI PRISM 377 DNA Sequencer For best results the PowerPlex Matrix Standards 3100 3130 Cat DG4650 should not be used to generate a matrix on the ABI PRISM 310 Genetic Analyzer The PowerPlex Matrix Standards 3100 3130 Cat DG4650 is required for spectral calibration on the ABI PRISM 3100 and 3100 Avant Genetic Analyzers and Applied Biosystems 3130 and 3130xl Genetic Analyzers The PowerPlex Matrix Standards 310 Cat DG4640 cannot be used to generate a matrix on these instruments For protocols and additional information on matrix standardization see the PowerPle
40. arams 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 IX D Store the size standard in the Size Standards folder at C AppliedBio Shared Analysis Sizecaller SizeStandards 7 Apply the size standard file to the samples then analyze the sample files See Section VI F for additional information on the use of the PowerTyper Y Macro Release 2 0 and Genotyper software Page 30 tmd018 0508 qxp 8 1 2008 3 25 PM Page 30 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD018 Revised 5 08 Page 31 Notes 1 Peak heights outside the linear range of the instrument may generate artifact peaks due to instrument saturation i e overloading the sample Bleedthrough pull ups from one color to another may be observed Saturated signal may 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 rela
41. at 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 radioactive silver stain or 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 studies as well The PowerPlex Y System a b allows co amplification and three color detection of twelve loci The system amplifies the loci DYS19 DYS385a b DYS389I II DYS390 DYS391 DYS392 DYS393 DYS437 DYS438 and DYS439 18 One primer specific for each of the DYS389I II 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
42. ch reaction contains the same master mix 3 Place one clean 0 2ml or 0 5ml reaction tube for each reaction into a rack and label appropriately Alternatively use a MicroAmp plate and label appropriately Note If using the GeneAmp PCR System 9600 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 4 Add the final volume of each reagent listed in Table 2 into a sterile 1 5ml amber colored tube Mix gently Table 2 shows the component volumes per reaction A worksheet to calculate the required amount of each PCR master mix component is provided in Section IX E 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 amplification yield than the larger loci Reducing the number of cycles in the amplification program by 2 to 4 cycles i e 10 20 or 10 18 cycling can improve locus to locus balance Page 8 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD018 Printed in USA Revised 5 08 Table 2 PCR Master Mix for the PowerPlex Y System PCR Master Mix Component1 Volume Per Reaction nuclease free water to a final volume of 25 0 l Gold ST R 10X Buffer 2
43. dder Mix and Internal Lane Standard 600 are light sensitive and must be stored in the dark We strongly recommend that pre amplification and postamplification reagents be stored and used separately with different pipettes tube racks etc Page 4 tmd018 0508 qxp 8 1 2008 3 24 PM Page 4 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD018 Revised 5 08 Page 5 Available Separately Product Size Cat 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 geneticidtools The proper panel and bin files for use with GeneMapper ID software can be obtained from the Promega web site at www promega com geneticidtools panels_bins Matrix standards are required for initial setup of the color separation matrix The matrix standards are sold separately and are available for the ABI PRISM 310 Genetic Analyzer and 377 DNA Sequencer PowerPlex Matrix Standards 310 and the ABI PRISM 3100 and 3100 Avant Genetic Analyzers and Applied Biosystem
44. ding the sample 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 VI F Sample Analysis Using the Genotyper Software and PowerTyper Y Macro To facilitate analysis of data generated with the PowerPlex 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 program and analyzed using the PowerTyper Y Macro Release 2 0 The PowerTyper Y Macro Release 2 0 is available upon request from Promega The PowerTyper Y Macro Release 2 0 is provided on the PowerTyper Macros CD ROM Cat DG3470 The PowerTyper Macros can b
45. e samples For the ABI PRISM 3100 and 3100 Avant Genetic Analyzers and Applied Biosystems 3130 and 3130xl Genetic Analyzers perform a new spectral calibration and re run the samples Instrument sensitivities can vary Optimize the injection or gel loading conditions See Section V 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 Page 40 tmd018 0508 qxp 8 1 2008 3 25 PM Page 40 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD018 Revised 5 08 Page 41 Symptoms Causes and Comments Allelic ladder not running Allelic ladder and primer pair mix were not compatible Ensure the same as samples that the allelic ladder is from the same kit as the primer pair mix Buffer incompatibility Samples were diluted in the wrong buffer Use Gold ST R 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 d
46. e also downloaded from the Promega web site at www promega com geneticidtools 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 from the PowerTyper Macros CD ROM Cat DG3470 to a designated location on your computer hard drive Alternatively download the PowerTyper Y Macro Release 2 0 from the Promega web site 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 tmd018 0508 qxp 8 1 2008 3 25 PM Page 33 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 TMD0
47. e colors This information must be entered to successfully analyze data with the PowerTyper Y Macro Release 2 0 8 In the BioLIMS Project column select 3100_Project1 from the pull down menu 9 In the Dye Set column select Z from the pull down menu 10 When using the ABI PRISM 3100 data collection software version 1 0 1 or 1 1 select GeneScan36_POP4PowerPlexY_3kV_11secs_2000 from the pull down menu in the Run Module 1 column Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD018 Revised 5 08 tmd018 0508 qxp 8 1 2008 3 24 PM Page 15 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 Revised 5 08 V B Detection of Amplified Fragments Using the ABI PRISM 3100 Genetic Analyzer with Data Collection Software Version 1 0 1 or 1 1 continued 11 To collect 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
48. eat 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 32 33 occurs when Taq DNA polymerase adds a nucleotide generally adenine to the 3 ends of amplified DNA fragments in a template independent manner The efficiency with which this occurs varies with different primer sequences Thus an artifact band one base shorter than expected i e missing the terminal addition is sometimes seen We have modified primer sequences and added a final extension step of 60 C for 30 minutes 34 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 35 36 Table 5 The PowerPlex Y System Allelic Ladder Information STR Locus Label Size Range of Allelic Ladder Components1 bases Repeat Numbers of Allelic Ladder Components Alleles Observed in 9948 Male DNA Positive Control2 DYS391 FL 90 118 6 8 13 10 DYS389I FL 148 168 10 15 13 DYS439 FL 203 231 8 153 12
49. ecan Freedom EVO 100 Profiles in DNA 9 1 8 10 42 Bjerke M et al 2006 Forensic application of the Maxwell 16 Instrument Profiles in DNA 9 1 3 5 43 Mandrekar P et al 2007 Introduction to Maxwell 16 low elution volume configuration for forensic casework Profiles in DNA 10 2 10 12 Additional STR references can be found at www promega com geneticidentity IX Appendix IX A Advantages of STR Typing STR typing is more tolerant of degraded DNA templates than other typing methods because amplification products are less than 500bp long much smaller than material detected using AMP FLP 23 or VNTR 24 analysis STR typing is also amenable to a variety of rapid DNA purification techniques which are compatible with PCR but do not provide enough DNA of appropriate quality for Southern blot based analyses Amplification products generated with Promega STR products are generally of discrete and separable lengths This allows construction of allelic ladders containing fragments of the same lengths as several or all known alleles for each locus Visual or software based comparison between the allelic ladder and amplified samples of the same locus allows rapid and precise assignment of alleles Results obtained using the PowerPlex Y System can be recorded in a digitized format allowing direct comparison with stored databases Population analyses do not require the use of arbitrarily defined fixed bins for populat
50. ect the run module you created in the previous step in the Run Module drop down list Lastly select F in the Dye Set drop down list Select OK Page 12 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD018 Printed in USA Revised 5 08 tmd018 0508 qxp 8 1 2008 3 24 PM Page 12 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD018 Revised 5 08 Page 13 3 In the Plate Manager create a new plate record as described in the instrument user s manual In the dialog box that appears select GeneMapper Generic in the Application drop down list and select the appropriate plate type 96 well Add entries in the owner and operator windows and select OK Note If autoanalysis of sample data is desired refer to the instrument user s manual for instructions 4 In the GeneMapper plate record enter sample names in the appropriate cells Scroll to the right In the Results group 1 column select the desired results group In the Instrument Protocol 1 column select the protocol you created in Step 2 Be sure this information is present for each row that contains a sample name Select OK Note To create a new results group select
51. ene mapping Science 235 1616 22 25 Budowle B and Monson K L 1989 In Proceedings of an International Symposium on the Forensic Aspects of DNA Analysis Government Printing Office Washington DC 26 Ayub Q et al 2000 Identification and characterisation of novel human Y chromosomal microsatellites from sequence database information Nucl Acids Res 28 e8 27 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 28 Gill P et al 1997 Considerations from the European DNA Profiling Group EDNAP concerning STR nomenclature Forensic Sci Int 87 185 92 29 Fr geau C J et al 1995 Characterization of human lymphoid cell lines GM9947 and GM9948 as intra and interlaboratory reference standards for DNA typing Genomics 28 184 97 30 Levinson G and Gutman G A 1987 Slipped strand mispairing A major mechanism for DNA sequence evolution Mol Biol Evol 4 203 21 31 Schlotterer C and Tautz D 1992 Slippage synthesis of simple sequence DNA Nucl Acids Res 20 211 5 32 Smith J R et al 1995 Approach to genotyping errors caused by nontemplated nucleotide addition by Taq DNA polymerase Genome Res 5 312 7 33 Magnuson V L et al 1996 Substrate nucleotide determined non templated addition of adenine by Taq DNA polymerase Implications for PCR based geno
52. ernal lane standard used in the loading cocktail can be increased or decreased to adjust the intensity of the size standard peaks The optimal peak height for the 100 base fragment of the internal lane standard is 500 1 000RFU If peak heights are too low we recommend altering the formamide internal lane standard mix to contain 1 0 l of ILS 600 and 9 0 l of Hi Di formamide If peak heights are too high we recommend altering the loading cocktail to contain 0 25 l of ILS 600 and 9 75 l of formamide 2 Mix for 10 15 seconds using a vortex mixer 3 Pipet 10 l of formamide internal lane standard mix into each well 4 Add 1 l of amplified sample or 1 l of allelic ladder mix Cover wells with appropriate septa Note Instrument detection limits vary therefore injection time or the amount of product mixed with loading cocktail may need to be increased or decreased Use the Module Editor in the Tools menu to modify injection time or voltage in the run module If peak heights are higher than desired Page 14 tmd018 0508 qxp 8 1 2008 3 24 PM Page 14 Page 15 samples can be diluted in Gold ST R 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
53. f amplified sample Note Instrument detection limits vary therefore the amount of product mixed with loading cocktail may need to be increased or decreased If peak heights are higher than desired samples can be diluted in Gold ST R 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 4 Combine 2 0 l of prepared loading cocktail and 1 0 l of PowerPlex Y Allelic Ladder Mix Vortex the allelic ladder mix prior to pipetting 5 Briefly centrifuge samples to bring the contents to the bottom of the tubes 6 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 7 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 8 Use a 60cc syringe filled with buffer and fitted with a bent 18 gauge needle to flush urea from the well area 9 Load 1 5 l 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
54. hile 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 Figure 2 The ramp rates for the GeneAmp PCR System 9700 thermal cycler 7486MB 94 0 C 100 90 0 C 100 60 0 C 29 58 0 C 29 70 0 C 23 70 0 C 23 3 tmp 10 cycles 3 tmp 22 cycles tmd018 0508 qxp 8 1 2008 3 24 PM Page 10 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD018 Revised 5 08 Page 11 V Instrument Setup and Sample Preparation V A Detection of Amplified Fragments Using the ABI PRISM 3100 or 3100 Avant Genetic Analyzer with Data Collection Software Version 2 0 and the Applied Biosystems 3130 or 3130xl Genetic Analyzer with Data Collection Software Version 3 0 Materials to Be Supplied by the User 95 C dry heating block water bath or thermal cycler crushed ice or ice water bath aeroso
55. ified with less than 32 cycles may work best with longer injection times 5 seconds Use of highly sensitive instrumentation 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 5 Select the appropriate matrix file Section III B 6 To analyze data automatically select the auto analyze checkbox and the appropriate analysis parameters and size standard Refer to the ABI PRISM 310 Genetic Analyzer User s Manual for specific information on these options 7 After loading the sample tray and closing the doors select Run to start the capillary electrophoresis system 8 Monitor electrophoresis by observing the raw data and status windows Each sample will take approximately 35 minutes for syringe pumping sample injection and sample electrophoresis Page 18 tmd018 0508 qxp 8 1 2008 3 24 PM Page 18 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD018 Revised 5 08 Page 19 V D Detection of Amplified Fragments
56. iliarize themselves with proper operation of the software For GeneMapper ID software version 3 1 users we recommend upgrading to version 3 2 Getting Started 1 Obtain the proper panel and bin files for use with GeneMapper ID from the Promega web site at www promega com geneticidtools panels_bins 2 Enter your contact information and select GeneMapper ID version 3 2 Select Submit 3 Select the PowerPlex Panels amp Bin Sets link and save the zip file to your computer 4 Open the files using the Windows WinZip program and save the unzipped files to a known location on your computer 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 08 tmd018 0508 qxp 8 1 2008 3 24 PM Page 23 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD018 Printed in USA Revised 5 08 Importing Panel and Bin Files For detailed instructions see the Applied Biosystems GeneMapper ID software tutorial 1 Open the GeneMapper ID software version 3 2 2 Select Tools then Panel Manager 3 Highlight the Panel Manager icon in the upper left tile navigation pane 4 Select File then Import Panels
57. imer Pair Mix 2 5 l AmpliTaq Gold DNA polymerase1 0 55 l 2 75u nuclease free water2 l Per tube template DNA volume2 0 25 1ng up to 19 45 l total reaction volume 25 l 1Assumes the AmpliTaq Gold DNA polymerase is at 5u l If the enzyme concentration is different the volume of enzyme must be adjusted accordingly 2The master mix volume and template DNA volume should total 25 l Consider the volume of template DNA and add nuclease free water to the master mix to bring the final volume of the final reaction to 25 l Figure 13 Internal Lane Standard 600 An electropherogram showing the Internal Lane Standard 600 fragments 5751TA 1 200 1 000 800 600 400 200 0 60 80 100 120 140 160 180 200 225 250 275 300 325 350 375 400 425 450 475 500 550 600 tmd018 0508 qxp 8 1 2008 3 25 PM Page 52 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD018 Revised 5 08 Page 53 IX F Composition of Buffers and Solutions IX G Related Products Fluorescent STR Multiplex Systems Product Size Cat PowerPlex 16 System 100 reactions DC6531 400 reactions DC6530 PowerPlex 16 BIO System 100 reactions DC6541 400 reactions DC6540 PowerPlex ES System
58. ing 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 Page 38 tmd018 0508 qxp 8 1 2008 3 25 PM Page 38 Schneider sequences The Schneider primer sequences are commonly used 13 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 VII Troubleshooting For questions not addressed here please contact your local Promega Branch Office or Distributor Contact information available at www promega com
59. ion data 25 Page 48 tmd018 0508 qxp 8 1 2008 3 25 PM Page 48 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD018 Revised 5 08 Page 49 IX B Advantages of Using the Loci in the PowerPlex Y System The loci included in the PowerPlex Y System Tables 4 and 5 have been selected because they represent well characterized loci generally accepted for forensic use This multiplex includes all loci in the European minimal haplotype DYS19 DYS385a b DYS389I II DYS390 DYS391 DYS392 and DYS393 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 9948 Male DNA standard template Table 4 The PowerPlex Y System Locus Specific Information STR Locus Label Chromosomal Location GenBank Accession Number Repeat Sequence1 5 3 DYS391 FL Yq G09613 TCTA 14 DYS389I II FL Yq AF140635 TCTG TCTA Complex 14 DYS439 FL Yq AC002992 GATA 26 DYS393 TMR Yq G09601 AGAT
60. ion of separated material Figure 1 Protocols for operation of the fluorescence detection instruments should be obtained from the instrument manufacturer Information on other Promega fluorescent STR systems and detection of amplified STR fragments using silver staining is available upon request from Promega or online at www promega com Amplification Setup Thermal Cycling Instrument Setup and Sample Preparation Data Analysis Section IV B Section V Section VI Section IV A GeneAmp PCR System 9700 GeneAmp PCR System 9600 GeneAmp PCR System 2400 Model 480 Thermal Cycler Applied Biosystems 3130 or 3130xl Genetic Analyzer with Data Collection Software Version 3 0 Section V A GeneMapper ID Software Versions 3 1 and 3 2 GeneScan Software and PC Operating Systems GeneScan Software and Macintosh Operating Systems ABI PRISM 3100 or 3100 Avant Genetic Analyzer with Data Collection Software Version 2 0 Section V A ABI PRISM 3100 Genetic Analyzer with Data Collection Software Version 1 0 1 or 1 1 Section V B ABI PRISM 310 Genetic Analyzer Section V C Figure 1 An overview of the PowerPlex Y System protocol ABI PRISM 377 DNA Sequencer Section V D tmd018 0508 qxp 8 1 2008 3 23 PM Page 3 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD018
61. l all ladders in the project are used If an allelic ladder fails to be analyzed or if many off ladder alleles are found in the samples samples should be re analyzed using another ladder from the project Page 34 tmd018 0508 qxp 8 1 2008 3 25 PM Page 34 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD018 Revised 5 08 Page 35 7 Double click on the Display Fluorescein Data macro to display the blue dye for all sample injections lanes Scroll down to observe and edit as needed 8 Double click on the Display TMR Data macro to display the yellow dye for all sample injections lanes Scroll down to observe and edit as needed 9 Double click on the Display JOE Data macro to display the green dye for all sample injections lanes Scroll down to observe and edit as needed 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 categorie
62. l resistant pipette tips 3100 or 3130 capillary array 36cm performance optimized polymer 4 POP 4 for the 3100 or 3130 10X genetic analyzer buffer with EDTA MicroAmp optical 96 well plate and septa Hi Di formamide Applied Biosystems Cat 4311320 PowerPlex Matrix Standards 3100 3130 Cat DG4650 The quality of formamide is critical Use Hi Di formamide with a conductivity less than 100 S cm Freeze formamide in aliquots at 20 C Multiple freeze thaw cycles or long term storage at 4 C may cause a breakdown of the formamide Formamide with a conductivity greater than 100 S cm may contain ions that compete with DNA during injection which results in lower peak heights and reduced sensitivity A longer injection time may not increase the signal Caution Formamide is an irritant and a teratogen avoid inhalation and contact with skin Read the warning label and take appropriate precautions when handling this substance Always wear gloves and safety glasses when working with formamide Sample Preparation 1 Prepare a loading cocktail by combining and mixing Internal Lane Standard 600 and Hi Di formamide as follows 0 5 l ILS 600 injections 9 5 l Hi Di formamide injections Note The volume of internal lane standard used in the loading cocktail can be increased or decreased to adjust the intensity of the size standard peaks The optimal peak height for the 10
63. lean 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 between 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 tmd018 0508 qxp 8 1 2008 3 24 PM Page 19 V D Detection of Amplified Fragments Using the ABI PRISM 377 DNA Sequencer continued 3 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 4 Filter the acrylamide solution through a 0 2 micron filter e g Nalgene tissue culture filter and degas for 5 minutes 5 Add 35 l of TEMED and 250 l of fresh 10 ammonium persulfate to 50ml of acrylamide solution and mix gently 6 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 to assist movement of solution to the bottom of the plates and to prevent formation of bubbles 7 Insert a 36 well sharkstooth comb or 34 well square tooth comb between the glass plates Sharkstooth combs
64. lex Matrix Standards 310 50 l each dye DG4640 PowerPlex Matrix Standards 3100 3130 25 l each dye DG4650 PowerTyper Macros 1 CD ROM DG3470 9948 Male DNA 250ng DD2061 9947A DNA 250ng DD1001 Internal Lane Standard 600 150 l DG1071 Gold ST R 10X Buffer 1 2ml DM2411 Mineral Oil 12ml DY1151 Nuclease Free Water 50ml 2 25ml P1193 Not for Medical Diagnostic Use For Laboratory Use Sample Preparation Systems Product Size Cat DNA IQ System 100 reactions DC6701 400 reactions DC6700 Differex System 50 samples DC6801 200 samples DC6800 Maxwell 16 Instrument each AS2000 DNA IQ Reference Sample Kit for Maxwell 16 48 preps AS1040 DNA IQ Casework Sample Kit for Maxwell 16 48 preps AS1210 Plexor HY System 800 reactions DC1000 200 reactions DC1001 Slicprep 96 Device 10 pack V1391 Not for Medical Diagnostic Use For Laboratory Use For Research Use Only Not for use in diagnostic procedures Polyacrylamide Gel Electrophoresis Reagents Product Size Cat Ammonium Persulfate 25g V3131 TBE Buffer 10X 1L V4251 Urea 1kg V3171 Blue Dextran Loading Solution 3ml DV4351 For Laboratory Use Page 54 tmd018 0508 qxp 8 1 2008 3 25 PM Page 54 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608
65. lex are registered trademarks of Promega Corporation Differex DNA IQ PowerTyper and Slicprep are trademarks of Promega Corporation ABI PRISM GeneMapper GeneScan Genotyper and MicroAmp are registered trademarks of Applera Corporation AmpliTaq Gold and GeneAmp are registered trademarks of Roche Molecular Systems Inc ART is a registered trademark of Molecular Bio Products Inc Biomek is a registered trademark of Beckman Coulter Inc Freedom EVO is a registered trademark of Tecan AG Corporation FTA is a registered trademark of Flinders Technologies Pty Ltd and is licensed to Whatman GenBank is a registered trademark of the U S Dept of Health and Human Services 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 Corporation Products may be covered by pending or issued patents or may have certain limitations Please visit our Web site for more information All prices and specifications are subject to change
66. ll cause your sizing quality to be flagged as red and no allele sizes will be called 5686TA Figure 12 An example showing improper assignment of size standard fragments in the GeneMapper ID software tmd018 0508 qxp 8 1 2008 3 25 PM Page 43 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 Revised 5 08 VII B GeneMapper ID Analysis Software continued Symptoms Causes and Comments Error message The bin set assigned to the analysis method may have been Both the Bin Set used in the deleted In the GeneMapper Manager select the Analysis Analysis Method and the Panel Methods tab and open the analysis method of interest Select must belong to the same the Alleles tab and select an appropriate bin set Chemistry Kit The wrong bin set was chosen in the analysis method Allele tab Be sure to choose the appropriate bin set as shown in Figure 3 Significantly raised baseline Poor spectral calibration for the ABI PRISM 3100 and 3100 Avant Genetic Analyzers and Applied Biosystems 3130 and 3130xl Genetic Analyzers Perform a new spectral calibration and re run the samples Poor matrix for the ABI PRISM 310 Genetic Analyzer Re run and optimize the matrix Use of Classic mode analysis method Use of Classic mode analysis on samples can result in baseline
67. md018 0508 qxp 8 1 2008 3 24 PM Page 24 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 08 Page 25 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 the 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 also change these settings 13 Select OK to save your settings 6361TA Figure 3 The Allele tab Select the bin set Promega_Bins_ID3 2 X tx
68. mported For Genotyper software versions 2 5 and 3 5 or higher set the preferences in the Edit menu to import the blue green yellow and red colors Error message Peak heights for one or more alleles in the allelic ladder Could not complete the sample file were below 150RFU The allelic ladder categories Run Macro command are defined as having a minimum peak height of 150RFU If because the labeled peak peak heights of ladder alleles are below 150RFU the software could not be found will not be able to locate the allele peak Re run the allelic ladder using more sample or longer injection time to assure peak heights above 150RFU CE spikes in the allelic ladder sample were identified as alleles by the macro Use a different injection of allelic ladder Allelic ladder data were not compatible with the PowerTyper file used Confirm that the PowerTyper Macro file matches the allelic ladder being used The base pair size of alleles in the allelic ladder are outside of the defined category range Be sure internal lane standard fragments are correctly sized Redefine internal lane standard fragments and re analyze the sample using GeneScan software Compare the size of the smallest allele in the allelic ladder with the base pair size and range listed in the categories for the same alleles If necessary increase the category start range in the category window to greater than 6bp and save the macro under a new name
69. ms 1 5ml amber colored microcentrifuge tubes Fisher Cat 05 402 26 aerosol resistant pipette tips see Section IX G 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 25 l reaction volume using the protocols detailed below Preferential amplification of smaller loci can occur 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 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 prior to multiplex analysis may provide some information as to the ratio of male to female DNA Amelogenin is a
70. nated as such for proper genotyping 3 In the Analysis Method column select the analysis method created in the Creating a Databasing or Paternity Analysis Method section 4 In the Panel column select PowerPlex_Y_ID3 2 X where X refers to the most recent version of the panel files This is the panel set that was imported in Section VI A 5 In the Size Standard column select the size standard that was created in the Creating a Size Standard section 6 If analyzing data from an ABI PRISM 310 Genetic Analyzer 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 VI D Sample Analysis Using the GeneScan Software and PC Operating Systems 1 Analyze data using the GeneScan analysis software 2 Review the raw data for one or more sample runs Highlight the sample file name then in the Sample menu select raw data Move the cursor so the crosshair is on the baseline to the right of the large primer peak before the first internal lane standard peak red Use the X value number shown at the bottom left of the window for the start position in the analysis parameters 3 The recommended analysis parameters are shown in Figure 8 4 The analysis parameters can be saved in the Params folder in most installations this is located at C AppliedBio Shared Analysis Sizecaller P
71. ne standard fragments are assigned correctly Re analyze the sample using GeneScan software and redefine the internal lane standard fragments VIII References 1 Edwards A et al 1991 DNA typing with trimeric and tetrameric tandem repeats Polymorphic loci detection systems and population genetics In The Second International Symposium on Human Identification 1991 Promega Corporation 31 52 2 Edwards A et al 1991 DNA typing and genetic mapping with trimeric and tetrameric tandem repeats Am J Hum Genet 49 746 56 3 Edwards A et al 1992 Genetic variation at five trimeric and tetrameric tandem repeat loci in four human population groups Genomics 12 241 53 4 Warne D et al 1991 Tetranucleotide repeat polymorphism at the human actin related pseudogene 2 actbp2 detected using the polymerase chain reaction 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
72. ntrol DNA allelic repeat sizes with the locus specific allelic ladder The expected 9948 DNA allele designations for each locus are listed in Table 5 Section IX B VI H Results Representative results of the PowerPlex Y System are shown in Figure 9 The PowerPlex Y Allelic Ladder Mix is shown in Figure 10 Page 36 6360TA A B C Figure 9 The PowerPlex Y System A single 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 tmd018 0508 qxp 8 1 2008 3 25 PM Page 36 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD018 Revised 5 08 Page 37 6371TA 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
73. oading the gel or capillary Artifacts of STR amplification PCR amplification of STR systems sometimes generates artifacts that appear as faint peaks one repeat unit smaller than the allele Stutter band peak heights can be high if the samples are overloaded See Section VI H for additional information on stutter and artifacts High background Load less amplification product or decrease injection time See Section V 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 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 th
74. of 1 0 2 0 l 10 Place the lid on the upper buffer chamber and close the instrument door Page 22 tmd018 0508 qxp 8 1 2008 3 24 PM Page 22 Page 23 Gel Electrophoresis and Detection 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 electrophoresis 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 HCl for 15 minutes then rinse thoroughly to remove any buildup VI Data Analysis VI A PowerPlex Panel and Bin Sets with GeneMapper ID Software Version 3 2 To facilitate analysis of data generated with the PowerPlex Y System we have created panel and bin files to allow automatic assignment of genotypes using GeneMapper ID software version 3 2 We recommend that users of GeneMapper ID software version 3 2 complete the Applied Biosystems GeneMapper ID Software Human Identification Analysis Tutorial to fam
75. plate 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 100X that of the male component can decrease the relative yield of some loci tmd018 0508 qxp 8 1 2008 3 25 PM Page 41 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD018 Printed in USA Revised 5 08 VII B GeneMapper ID Analysis Software Symptoms Causes and Comments Alleles not called To analyze samples with GeneMapper ID software the analysis parameters and size standard must both have Basic or Advanced as the analysis type If they are different an error is obtained Figure 11 An insufficient number of ILS 600 fragments was defined Be sure to define at least one ILS 600 fragment smaller than the smallest sample peak and at least one ILS 600 fragment larger than the largest sample peak Run was too short and larger peaks in ILS were not captured Not all ILS 600 peaks defined in the size standard were detected during the run Create a new size standard using the internal lane standard fragments present in the sample Re run samples using a longer run time Off ladder alleles An allelic ladder from a different run than the samples was used Re analyze the samples with an allelic ladder from the same run The GeneMapper
76. s 3130 and 3130xl Genetic Analyzers PowerPlex Matrix Standards 3100 3130 See Section IX G for ordering information III Before You Begin III 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 as well as small changes in buffers ionic strength primer concentrations choice of thermal cycler and thermal cycling conditions can affect PCR success We suggest strict adherence to recommended procedures for amplification as well as denaturing gel electrophoresis and fluorescence detection PCR based STR analysis is subject to contamination by very small amounts of nontemplate 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 ST R 10X Buffer and PowerPlex Y 10X Primer Pair Mix are provided in a separate box and 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 IX G Some reagents used in the an
77. s loci in columns while the Vertical table format displays the categories in rows These tables can be customized to fit needs To save data in tables go to the Table drop down menu highlight Export to File and save the file with the desired name and location The saved file can be viewed and analyzed using Microsoft Excel PowerTable Format Allele Table Format Vertical Table Format 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 Sample Info Category Peak 1 Peak 2 Sample Info Sample Comment Category Peak 1 Peak 2 Peak 3 Peak 4 Over flow Low Signal Satura tion Edited Label Edited Row Sample Info Category Allele 1 Category Allele 2 Category Allele 1 Category Allele 2 Category Allele 1 Category Allele 2 Category Allele 1 Category Allele 2 tmd018 0508 qxp 8 1 2008 3 25 PM Page 35 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 Revised 5 08 VI G Controls 1 Observe the results for the negative control The negative control should be devoid of amplification products 2 Observe the results for the 9948 Male DNA positive control Compare the co
78. s with more noise than those analyzed using the Basic or Advanced mode analysis method Advanced mode analysis methods and size standards are recommended Red bar appears during analysis If none of the samples had matrices applied when run on the of samples and the following ABI PRISM 310 Genetic Analyzer no data will be displayed error message appears when Apply a matrix file during analysis in the GeneMapper ID data are displayed Some selected software and re analyze sample s do not contain analysis data Those sample s will not be shown Error message after attempting There was a conflict between different sets of panel and bin to import panel and bin files files Delete all panel and bin sets and re import files in a Unable to save panel data different order java SQLEException ORA 00001 unique constraint IFA CKP_NNN violated Allelic ladder peaks are GeneMapper ID software was not used or microsatellite labeled off ladder OL 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
79. t where X refers to the most recent version of the bin set tmd018 0508 qxp 8 1 2008 3 24 PM Page 25 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 Revised 5 08 VI B Creating a Casework Analysis Method with GeneMapper ID Software continued Creating a Size Standard 1 Select Tools then GeneMapper Manager 2 Select the Size Standard tab 3 Select New 4 Select Basic or Advanced Figure 5 The type of analysis method selected must match the type of analysis method created earlier Select OK 5 Enter a detailed name such as ILS 600 advanced in the Size Standard Editor Figure 6 6 Choose red as the color for the size standard dye 7 Enter the sizes of the internal lane standard fragments see Section IX D Figure 13 8 Select OK Page 26 5723TA Figure 4 The Peak Detector tab tmd018 0508 qxp 8 1 2008 3 24 PM Page 26 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD018 Revised 5 08 Page 27 5726TA Figure 6 The Size Standard Editor 5725TA Figure 5 The Select Dye and Analysis Method window tmd018 0508 qxp 8 1 2008 3 24 PM Page 27
80. tains 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 V 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 tmd018 0508 qxp 8 1 2008 3 25 PM Page 51 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 Revised 5 08 Page 52 IX E Preparing the PowerPlex Y System Master Mix A worksheet to calculate the required amount of each PCR master mix component is provided in Table 6 Multiply the volume l per reaction by the total number of reactions to obtain the final master mix volume l Table 6 Master Mix for the PowerPlex Y System PCR Master Mix Component Volume Per Reaction Number of Reactions Final Volume l Gold ST R 10X Buffer 2 5 l PowerPlex Y 10X Pr
81. the ABI PRISM 310 data collection software 2 Prepare a GeneScan sample sheet as described in the ABI PRISM 310 Genetic Analyzer User s Manual Enter the appropriate sample information in the sample info column For rows containing PowerPlex 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 tmd018 0508 qxp 8 1 2008 3 24 PM Page 17 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 Revised 5 08 V C Detection of Amplified Fragments Using the ABI PRISM 310 Genetic Analyzer continued 3 Create a new GeneScan injection list Select the appropriate sample sheet by using the pull down menu 4 Select the GS STR POP4 1ml A Module using the pull down menu Change the injection time to 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 ampl
82. tive 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 6054TA Figure 8 The Analysis Parameters window The start point of the analysis range which will vary is defined in Section VI D Step 2 tmd018 0508 qxp 8 1 2008 3 25 PM Page 31 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 Revised 5 08 VI E Sample Analysis Using the GeneScan Software and Macintosh Operating Systems 1 Analyze data using the GeneScan analysis software 2 Review the raw data for one or more sample runs Highlight the sample file name then in the Sample menu select raw data Move the cursor so the crosshair is on the baseline to the right of the large primer peak before the first internal lane standard peak red Use the X value number shown at the bottom left of the window for the start position in the analysis parameters 3 The recommended analysis parameters are 4 The analysis parameters can be saved in the Params folder 5 Apply the stored analysis parameters file to the samples 6 Assign a new size standard Select a sample file highlight the arrow next to size standard then select define new Assign the size standard peaks as shown
83. tively 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 yield 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 reduc
84. ty Analysis Method with GeneMapper ID Software 1 Select Tools then GeneMapper Manager 2 Select the Analysis Methods tab 3 Select New and a new analysis method dialog box will open 4 Select HID and select OK Note If you do not see the HID option you do not have the GeneMapper ID software Contact Promega Technical Services by e mail genetic promega com for assistance 5 Enter a descriptive name for the analysis method such as PowerPlexY_20 filter 6 Select the Allele tab 7 Select the bin set corresponding to the PowerPlex System Promega_Bins_ID3 2 X where X refers to the most recent version of the bin set 8 Ensure that the Use marker specific stutter ratio if available box is checked Page 28 tmd018 0508 qxp 8 1 2008 3 24 PM Page 28 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD018 Revised 5 08 Page 29 9 Enter the values shown in Figure 7 for proper filtering of peaks when using the PowerPlex Y System For an explanation of the proper usage and effect of these settings refer to the Applied Biosystems user bulletin titled Installation Procedures and New Features for GeneMapper ID Software 3 2 Creating a Size Standard 1 Select Tools then GeneMapper Manager
85. typing BioTechniques 21 700 9 34 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 tmd018 0508 qxp 8 1 2008 3 25 PM Page 47 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD018 Printed in USA Revised 5 08 VIII References continued 35 Moller A Meyer E and Brinkmann B 1994 Different types of structural variation in STRs HumFES FPS HumVWA and HumD21S11 Int J Leg Med 106 319 23 36 Brinkmann B Moller A and Wiegand P 1995 Structure of new mutations in 2 STR systems Int J Leg Med 107 201 3 37 Mandrekar P V Krenke B E and Tereba A 2001 DNA IQ The intelligent way to purify DNA Profiles in DNA 4 3 16 38 Krenke B E et al 2005 Development of a novel fluorescent two primer approach to quantitative PCR Profiles in DNA 8 1 3 5 39 Greenspoon S and Ban J 2002 Robotic extraction of sexual assault samples using the Biomek 2000 and the DNA IQ System Profiles in DNA 5 1 3 5 40 McLaren B Bjerke M and Tereba A 2006 Automating the DNA IQ System on the Biomek 3000 Laboratory Automation Workstation Profiles in DNA 9 1 11 13 41 Cowan C 2006 The DNA IQ System on the T
86. ue 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 Use of FTA paper Results may be similar to those obtained with excess amounts of male DNA template Reduce the number of cycles in the amplification program by 2 4 cycles 10 20 or 10 18 cycling to improve locus to locus balance Degraded DNA sample DNA template is degraded and larger loci show diminished yield Repurify the template DNA Insufficient male template DNA Use the recommended amount of male template DNA Stochastic effects can occur when amplifying low amounts of template 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 tem
87. uffer 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 select 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 tmd018 0508 qxp 8 1 2008 3 24 PM Page 21 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 Revised 5 08 V D Detection of Amplified Fragments Using the ABI PRISM 377 DNA Sequencer continued Sample Preparation and Loading 1 Prepare a loading cocktail by combining and mixing ILS 600 and Blue Dextran Loading Solution as follows 0 5 l ILS 600 lanes 1 5 l Blue Dextran Loading Solution 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 2 Vortex for 10 15 seconds 3 Combine 2 0 l of prepared loading cocktail and 1 0 l o
88. vailable as a monoplex amplification with a ladder labeled with either fluorescein Cat DC5171 or TMR Cat DC6171 IV A Amplification Setup The use of gloves and aerosol resistant pipette tips is highly recommended to prevent cross contamination Keep all pre amplification and postamplification 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 VII A 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 Pair Mix as this may cause the primers to be concentrated at the bottom of the tube 2 A precipitate may form in the Gold ST R 10X Buffer If this occurs warm the solution briefly at 37 C then vortex until the precipitate is in solution tmd018 0508 qxp 8 1 2008 3 24 PM Page 7 IV A Amplification Setup continued 2 Determine the number of reactions to be set up This should include positive and negative control reactions Add 1 or 2 reactions to this number to compensate for pipetting error While this approach does waste a small amount of each reagent it ensures that you will have enough PCR master mix for all samples It also ensures that ea
89. was analyzed with GeneMapper ID software version 3 2 and PowerPlex Y panel and bin files Panel A The fluorescein labeled allelic ladder components and their allele designations Panel B The JOE labeled allelic ladder components and their allele designations Panel C The TMR labeled allelic ladder components and their allele designations tmd018 0508 qxp 8 1 2008 3 25 PM Page 37 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 Revised 5 08 VI H Results continued Stutter and Artifacts Stutter bands 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 products 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 respec
90. with 64 or 96 wells also may be used 8 Secure the comb with 3 evenly spaced clamps 9 Keep the remaining acrylamide solution as a polymerization control 10 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 Page 20 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD018 Printed in USA Revised 5 08 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 tmd018 0508 qxp 8 1 2008 3 24 PM Page 20 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD018 Revised 5 08 Page 21 Instrument Preparation 1 Open the ABI PRISM 377 data collection software 2 Prepare a sample sheet as described in the GeneScan Analysis Software User
91. 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 tmd018 0508 qxp 8 1 2008 3 25 PM Page 55
92. x 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 upon request from Promega or online at www promega com tbs Page 6 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 tmd018 0508 qxp 8 1 2008 3 24 PM Page 6 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD018 Revised 5 08 Page 7 IV Protocols for DNA Amplification Using the PowerPlex Y System Materials to Be Supplied by the User model 480 or GeneAmp PCR System 9600 9700 or 2400 thermal cycler Applied Biosystems microcentrifuge 0 5ml or 0 2ml thin walled microcentrifuge tubes or MicroAmp optical 96 well reaction plate Applied Biosyste
93. ycles in the amplification program by 2 4 cycles to achieve the desired signal intensity 5 Centrifuge plate briefly to remove air bubbles from the wells if necessary 6 Denature samples at 95 C for 3 minutes then immediately chill on crushed ice or in an ice water bath for 3 minutes Denature samples just prior to loading the instrument Instrument Preparation Refer to the instrument users manual for instructions on cleaning installing the capillary array performing a spatial calibration and adding polymer Analyze samples as described in the user s manual for the ABI PRISM 3100 or 3100 Avant Genetic Analyzer with data collection software version 2 0 and the Applied Biosystems 3130 or 3130xl Genetic Analyzer with the following exceptions 1 In the Module Manager select New Select Regular in the Type drop down list and select HIDFragmentAnalysis36_POP4 in the Template drop down list Confirm that the injection time is 5 seconds and the injection voltage is 3kV Lengthen the run time to 2 000 seconds Give a new name to your run module and select OK Note Instrument sensitivities can vary The injection time and voltage may be adjusted in the Module Manager A suggested range for the injection time is 3 22 seconds and for the injection voltage is 1 3kV 2 In the Protocol Manager select New Type a name for your protocol Select Regular in the Type drop down list and sel

PowerPlex® Y System

Contents

Download Pdf Manuals

Related Search

Related Contents

PowerPlex&reg; Y System

Contents

Download Pdf Manuals

Related Search

Related Contents

PowerPlex® Y System