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AmpFlSTR® NGM™ PCR Amplification Kit User`s Guide (PN

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1. E Reference Samples m n H Manager Maker Name Color Min Size Marker Marker Comments Ladder Alleles JAmpFLSTR_Panels_v1 POS1248 20 fzo 1215 4 0 1289 8 9 10 11 12 13 14 15 18 17 CHES AmpFLSTR_NGM_v2 WWA blue 214 3 14 16 4 0 1182 none 11 12 13 14 15 16 17 18 19 TS 0165539 blue 2776 340 4 01057 none X 1589404142434445 AWA D2S1338 bue 2816 3560 2023 4 0 1355 none 15 16 17 18 19 20 21 22 23 0165539 AMEL green 100 0 108 0 xy 9 0 0 KY 0251338 0851179 green 1179 1749 1213 4 0 1082 8 9 10 11 12 13 14 15 16 17 021511 green 2498 2831 4 0114 none 24 24 2 25 26 27 28 28 2 29 521544 D18551 green 2595 3475 12 15 4 0 1389 7 9 10 10 2 11 12 13 13 2 1 018551 02251045 yelow 760 1200 116 3 0 1799 none 8 9 10 11 12 13 14 15 16 17 02251045 0195433 yellow 1223 1663 445 4 0 1106 9 10 11 12 12 2 13 13 2 14 5 THOT yelow 1764 2211 793 4 00526 14567 89931011133 FGA 12 FGA yellow 2216 3720 2426 4 0 1261 17 18 19 20 21 22 23 24 25 D25441 13 025441 red 745 1134 Msas 4 0 0947 none 9 10 11 11 3 12 13 14 15 16 0351358 14 10351358 red 1144 1684 5 16 4 0 1377 12 13 14 15 16 17 18 19 15 10151656 1700
2. 98 B Chemical alerts 99 AmpF amp STR PCR Amplification Kit User s Guide 93 Appendix Safety Chemical safety Chemical hazard WARNING CHEMICAL HAZARD Before handling any chemicals refer warning to the Safety Data Sheet SDS provided by the manufacturer and observe all relevant precautions WARNING CHEMICAL HAZARD chemicals in the instrument including liquid in the lines are potentially hazardous Always determine what chemicals have been used in the instrument before changing reagents or instrument components Wear appropriate eyewear protective clothing and gloves when working on the instrument AN WARNING CHEMICAL STORAGE HAZARD Never collect or store waste in a glass container because of the risk of breaking or shattering Reagent and waste bottles can crack and leak Each waste bottle should be secured in a low density polyethylene safety container with the cover fastened and the handles locked in the upright position Wear appropriate eyewear clothing and gloves when handling reagent and waste bottles Chemical safety minimize the hazards of chemicals guidelines Read and understand the Safety Data Sheets SDSs provided by the chemical manufacturer before you store handle or work with any chemicals or hazardous materials See About SDSs on page 94 Minimize contact with chemicals We
3. software ibili Operating Data Collection compatibility Instrument system Software Analysis software 3500 3500xL Windows 3500 Series GeneMapper D X Software Windows Data Collection v1 2 Vista Software v1 0 3130 3130x Windows XP 3 0 GeneMapper ID Software v3 2 1 3100 3100 Windows NT 1 1 3100 and Avant 1 0 3100 Avant GeneMapper ID X Software v1 0 1 or higher Windows 2000 2 0 310 Windows XP 3 1 Windows NT and 3 0 Windows 2000 Applied Biosystems conducted validation studies for the AmpF STR NGM Kit using this configuration About Applied Biosystems fluorescent multi color dye technology allows the analysis of multicomponent multiple loci including loci that have alleles with overlapping size ranges Alleles for analysis overlapping loci are distinguished by labeling locus specific primers with different colored dyes Multicomponent analysis is the process that separates the 5 different fluorescent dye colors into distinct spectral components The 4 dyes used in the NGM PCR Amplification Kit to label samples are 6 FAM VIC NED and PET dyes The fifth dye LIZ is used to label the GeneScan 500 LIZ Size Standard AmpF amp STR PCR Amplification Kit User s Guide 15 Chapter 1 Overview How multicomponent 16 analysis works Each of these fluorescent dyes emits its maximum fluorescence at a different wavele
4. 23 B DNA 23 Prepare the amplification kit reactions llle else 25 B PerformePGR 5s tese dt ote e e tue tb utu tata 26 m Amplification using bloodstained FTA 27 AmpF amp STR PCR Amplification Kit User s Guide 21 Chapter 2 Amplification PCR work areas Work area setup and lab design PCR setup tools Many resources are available for the appropriate design of a PCR laboratory For AmpF STR NGM PCR Amplification Kit forensic DNA testing refer to National Institute of Justice Office of Law Enforcement Standards 1998 Forensic Laboratories Handbook for Facility Planning Design Construction and Moving Washington DC National Institute of Justice 76 pp For AmpF STR NGM Kit parentage DNA testing refer to American Association of Blood Banks 2004 Guidance for Standards for Parentage Relationship Testing Laboratories 7th ed Bethesda Md American Association of Blood Banks 58 pp The sensitivity of the AmpFLSTR NGM Kit and other PCR based tests enables amplification of minute quantities of DNA necessitating precautions to avoid contamination of samples yet to be amplified Kwok and Higuchi 1989 To prevent contamination by human DNA be careful while handling and processing samples Wear gloves at all times and change them frequently Close sample tubes wh
5. 0165539 Ratio From Distance To Distance Ratio From Distance To Distance 8 0251338 1 1755 2 25 3 75 1 70710 2 25 3 75 AMEL T 2 2 G 0851179 021511 3 018551 4 4 02251045 Stutter Ratio amp Dist tH 0195433 11 Click Apply then OK to add the AmpF STR NGM Kit panels bin sets and marker stutter to the GeneMapper Software database IMPORTANT If you close the Panel Manager without clicking Apply the panels bin sets and marker stutter will not be imported into the GeneMapper ID X Software database 68 PCR Amplification Kit User s Guide Set GeneMapper ID X Software for data analysis Create an Use the following procedure to create an analysis method for the AmpF STR analysis method NGM Kit IMPORTANT Analysis methods are version specific so you must create an analysis method for each version of the software For example an analysis method created for GeneMapper D X version 1 2 is not compatible with GeneMapper D X Software v1 0 v1 1 or with ID Software version 3 2 1 1 Select Tools GeneMapper 1D X Manager to open the GeneMapper D X Manager ID X Manager Find Name Containing Projects Analysis Methods Table Settings Plot Settings Matrices Size Standards Report Settings Name Last Saved Owner Instrument Analysis Type AmpFLSTR_Ana
6. Item Source MicroAmp 96 Well Full Plate Cover N8010550 MicroAmp 96 Well Tray Retainer Set 403081 POP 4 Polymer for the 310 Genetic Analyzer 402838 For a complete list of parts and accessories for the 310 instrument refer to Appendix of the ABI PRISM 310 Genetic Analyzer User Guide Part no 4317588 PCR Amplification MicroAmp 96 Well Tray N8010541 MicroAmp Reaction Tube with Cap 0 2 mL N8010540 MicroAmp 8 Tube Strip 0 2 mL N8010580 MicroAmp 8 Cap Strip N8010535 MicroAmp 96 Well Tray Retainer Set 403081 MicroAmp 96 Well Base N8010531 MicroAmp Clear Adhesive Film 4306311 MicroAmp Optical Adhesive Film 4311971 MicroAmp Optical 96 Well Reaction Plate N8010560 Other user supplied materials Hi Di Formamide 25 mL 4311320 Aerosol resistant pipette tips MLS Microcentrifuge tubes MLS Pipettors MLS Tape labeling MLS Tube 50 mL Falcon MLS Tube decapper autoclavable MLS Deionized water PCR grade MLS Tris HCL pH 8 0 MLS EDTA 0 5 M MLS Vortex MLS AmpF amp STR PCR Amplification Kit User s Guide 91 Appendix Ordering Information 92 AmpFSTR PCR Amplification Kit User s Guide Safety This appendix covers B Chemical safety a O odd aw aa 94 m Chemical waste 96 Biological hazard safety
7. Size Standard Dye TSize Standard Table Size in Basepairs i 100 0 139 0 150 0 160 0 200 0 300 0 350 0 400 0 O 22 lt o 12 o 2 O 2 2 450 0 AmpF amp STR PCR Amplification Kit User s Guide 75 Section 4 1 Data Analysis Analyze and edit sample files with GeneMapper D X Software Analyze a project 1 In the Project window select File gt Add Samples to Project then navigate to the disk or directory containing the sample files 2 Apply analysis settings to the samples in the project The names of the settings shown are the names suggested in the sections above If you named the settings differently select the name you specified Parameter Settings Sample Type Select the sample type Analysis Method NGM_AnalysisMethod_v2X Panel NGM panel v2X Size Standard CE G5 NGM GS500 e Size Standard For more information about how the Size Caller works refer to the ABI PRISM GeneScan Analysis Software for the Windows NT Operating System Overview of the Analysis Parameters and Size Caller User Bulletin Part no 4335617 CE_G5_NGM_GS500 size standard fragments defined in the AmpF STR NGM Kit 75 100 139 150 160 200 300 350 400 and 450 For additional information about size standards refer to the GeneMapper ID Software Version 3 1 Human Identification Analysis
8. gt Note If a sample allele peak is called as an off ladder allele verify sample result according to your laboratory s protocol AmpF amp STR PCR Amplification Kit User s Guide 63 Section 4 1 Data Analysis Set up GeneMapper ID X Software for data analysis 64 Workflow analyze sample fsa files using GeneMapper 0 Software v1 0 1 or higher for the first time e Import panels bins and marker stutter into the Panel Manager as explained in Import panels bins and marker stutter on page 64 Create an analysis method as explained in Create an analysis method on page 69 Create a size standard as explained in Create a size standard on page 74 Define custom views of analysis tables Refer to the GeneMapper ID X Software Version 1 0 Getting Started Guide Part no 4375574 for more information Define custom views of plots Refer to the GeneMapper ID X Software Version 1 0 Getting Started Guide Part no 4375574 for more information Import import the AmpF STR NGM Kit panels bin sets and marker stutter from the panels bins and Applied Biosystems web site into the GeneMapper D X Software database marker stutter 1 Download and open the file containing panels bins and marker stutter a From the Support menu of www appliedbiosystems com select Support gt Software Downloads Patches amp Updates GeneMapper ID X Softwar
9. 8 Select D10S1248 to display the Bin view for the marker in the right pane 66 AmpF amp STR PCR Amplification Kit User s Guide Set up GeneMapper ID X Software for data analysis Panel Manager File Edit Bins View Help Bin Set NGM bins v2X 4 stp Panel Manager E E AmpFLSTR_NGM _v2X j 0165539 D251338 AMEL j 0851179 D21511 D18551 02251045 0195433 01 FGA 025441 0351358 0151656 0125391 AmpFLSTR_Panels_v1X Reference Samples 0 0 67 69 71 73 75 7 81 83 85 87 89 91 93 95 97 99 101 103 105 107 109 111 113 115 117 119 121 123 125 127 129 131 21051248 9 Import NGM_stutter_v2X a Select the AmpFLSTR_NGM_v2X folder in the navigation panel Panel Manager File Edit Bins View Help BP bins Panel Name Comment S gf Panel Manager fft AmpFLSTR v2X AmpFLSTR Panels v1X 3M panel v2X null b Select File Import Marker Stutter to open the Import Marker Stutter dialog box c Navigate to then open the NGM Analysis Files GMIDX folder d Select NGML stutter v2X then click Import O 22 lt o 12 P O 2 B Note Importing this file associates the marker stutter ratio with the bin set in the NGM_bins_v2X folder AmpF amp STR PCR Amplification Kit User s
10. Analysis Method Editor General Allele Peak Detector Peak Quality SQ amp GQ Settings Bin Set NGM_bins_v2X Use marker specific stutter ratio and distance if available Marker Repeat Type Tri Tetra Penta Global Cut off Value 0 0 0 0 0 0 MinusA Ratio 0 0 0 0 0 0 MinusA Distance 0 0 0 0 0 0 0 0 0 0 0 0 Global Minus Stutter Ratio 0 0 0 0 0 0 Global Minus Stutter Distance From 2 25 0 0 To 0 0 Global Plus Stutter Ratio 0 0 Global Plus Stutter Distance From e T 0 0 To 0 0 Amelogenin Cutoff In the Bin Set field select NGM_bins_v2X bin set imported previously and configure the stutter distance parameters as shown GeneMapper D X Software allows you to specify 4 types of marker repeat motifs tri tetra penta and hexa You can enter parameter values for each type of repeat in the appropriate column The Use marker specific stutter ratio if applicable check box is selected by default When this box is checked the software applies the stutter ratio filters in the NGM_stutter_v2X file CD 22 lt 2 2 AmpF amp STR PCR Amplification Kit User s Guide 71 Section 4 1 Data Analysis 72 Peak Detector tab settings Analysis Method Editor General Allele Peak Detector Peak Qualit
11. IMPORTANT The fluorescent dyes attached to the primers are light sensitive Protect the AmpF STR NGM Primer Set from light when not in use Amplified DNA AmpFLSTR NGM Allelic Ladder and GeneScan 500 LIZ Size Standard should also be protected from light Keep freeze thaw cycles to a minimum requirements The following table lists Data Collection Software and the run modules that can be used to analyze AmpF STR NGM Kit PCR products For details on the procedures refer to the documents listed in the table Data Operating Collection System Run modules and conditions References Software 3 01 Windows e HIDFragmentAnalysis36_POP4_1 Applied Biosystems 3130 3130xl Genetic 3130 3130x Injection conditions Analyzers Using Data Collection Software Analyzer 3130 3 kV 5 v3 0 Protocols for Processing AmpF4STR SSF PCR Amplification Kit PCR Products User 3130x 3 kV 10 sec Bulletin Part no 4363787 Dye Set G5 2 0 Windows e HIDFragmentAnalysis36_POP4_1 ABI Pg SM 3100 3100 Avant Genetic 3100 2000 Injection condition 3kV 10 sec Analyzers Using Data Collection Software Analyzer D v2 0 Protocols for Processing AnpFSTR ye Set G5 PCR Amplification Kit PCR Products User Bulletin Part no 4350218 1 1 Windows e GeneScan36vb DyeSetG5Module ABI Prism 3100 3100 Avant Genetic 8100 NT Injection condition 3kV 10 sec Analyzers Protocols for Processing Analyzer e GS500Analvsi AmpF amp TR PCR A
12. Panel Manager File Edit Bins View Help uox NEN Panel Comment S gf Panel Manager AmpFLSTR_NGM_v2X H AmpFLSTR_Panels_v1X 3M panel 2 null b Select File Import Bin Set to open the Import Bin Set dialog box c Navigate to then open the NGM Analysis Files GMIDX folder d Select NGM bins v2X then click Import Note Importing this file associates the bin set with the panels in the panel v2X folder AmpF4STR9 PCR Amplification Kit User s Guide 65 O 22 lt D 2 2 Section 4 1 Data Analysis Import Bin Set NGM Analysis Files GMIDX 2 555 NGM_bins_v2X txt NGM panel v2X txt stutter v2X txt My Documents File name bins v2X txt ure 7 View the imported panels the navigation pane a Double click the AmpFLSTR_NGM_v2X folder to view the NGM_panel_v2X folder b Double click the NGM_panel_v2X folder to display the panel information in the right pane and the markers below it Panel Manager Edt Eins View Help ij X Bin Set v BER G S f Panel Manager Marker Name Dye Color Sim Size Control Alleles Marker Ladder Alleles AmpFLSTR_N
13. Kelsey Z Webb R Evans J and Gill P 1998 The development of a third generation STR multiplex system TGM Olaisen B Brinkmann B and Lincoln PJ eds Progress in Forensic Genetics 7 Proceedings of the 17th International ISFH Congress Oslo 2 6 September 1997 Elsevier Amsterdam pp 192 194 Weber J and Wong 1993 Mutation of human short tandem repeats Mol Genet 2 1123 1128 Wiegand P Schneider H R Schurenkamp M Kleiber M and Brinkmann B 1998 Tetranucleotide STR system D8S1132 sequencing data and population genetic comparisons nt J Legal Med 111 4 180 182 Wiegand P and Kleiber M 2001 Less is more length reduction of STR amplicons using redesigned primers Int J Legal Med 114 285 287 AmpFISTR NGM PCR Amplification Kit User s Guide Symbols fsa sample files 49 64 A allelic ladder about 18 figure 12 number per run suggested 34 requirements for accurate genotyping 34 volume per reaction 36 38 42 amplification amplified DNA 22 loci 11 using bloodstained FTA cards 27 work area tools 22 B biohazardous waste handling 98 CAUTION description 7 chemical safety 94 chemical waste safety 96 contents of kit 17 24 control DNA 007 13 17 D DANGER description 7 Data Collection Software 15 DNA amplified 22 control about 17 negative control reaction 25 positive control reaction 25 quantification methods 23 24 sample prepar
14. 16 2 17 17 2 THO1 11p15 5 4 5 6 7 8 9 9 3 10 11 13 3 NED 7 9 3 FGA 4q28 17 18 19 20 21 22 23 24 25 26 26 2 NED 24 26 27 28 29 30 30 2 31 2 32 2 33 2 42 2 43 2 44 2 45 2 46 2 47 2 48 2 50 2 51 2 02 441 2 14 9 10 11 11 3 12 13 14 15 16 14 15 03 1358 3 21 31 12 13 14 15 16 17 18 19 15 16 01 1656 1942 2 9 10 11 12 13 14 14 3 15 15 3 16 13 16 16 3 17 17 3 18 3 19 3 20 3 0125391 12 13 2 14 15 16 17 18 19 19 3 20 21 22 23 18 19 24 25 26 27 AmpF amp STR PCR Amplification Kit User s Guide 11 Chapter 7 Overview Allelic ladder Figure 1 shows the allelic ladder for the AmpF STR NGM Kit See Allelic ladder profile requirements on page 34 for information on ensuring accurate genotyping Allelic_Ladder_HO2_016 fsa Allelic_Ladder Mark Sample for Deletion 70 110 150 190 230 270 310 350 Lshsh7hisho po bi b2b3 pe bs pop be Allelic_Ladder_HO2_016 fsa Altelic Ladder Mark Sample for Deletion 70 110 150 130 230 270 310 350 1200 800 400 0 m M M a a n hbBEslEEchyhebobobib2bsbtbsbsb Allelic_Ladder_HO2_016 fsa Allel
15. 240 316 4 0 1416 9 10 11 12 13 14 14 3 15 15 16 ID12S391 red 2250 2870 1819 4 0 1584 14 15 16 17 18 19 19 3 20 2 8 Select D10S1248 to display the Bin view for the marker in the right pane AmpF amp STR PCR Amplification Kit User s Guide 51 O 5 0 o h P e Chapter 4 Data Analysis F Panel Manager File Edit Bins View m L amp E Panel Manager AmpFLSTR_Panels_v1 AmpFLSTR_NGM_v2 I A NGM panel v2 1 0165539 0251338 AMEL 0851179 D21811 018551 02251045 0195433 THO1 FGA 025441 0351358 0151656 0125391 E Reference Samples M safe m TR T T mim 14 15 16 17 18 71 75 21051248 115 119 123 127 131 52 Click Apply then OK to add the AmpF STR NGM panel and bin set to the GeneMapper ID Software database IMPORTANT If you close the Panel Manager without clicking OK the panels and bins are not imported into the GeneMapper JD Software database Create a HID analysis method The HID Advanced analysis method for the AmpF STR NGM Kit uses the NGM_bins_v2 file described in step 6 on page 50 Use the following procedure to create a HID analysis method for the AmpFISTR Kit 1 Select Tools GeneMapper Manager to open the GeneM
16. Applied Biosystems 3130 3130xl Genetic Analyzers Getting Started Guide 4352715 Applied Biosystems 3130 3130xl Genetic Analyzers Maintenance 4352716 Troubleshooting and Reference Guide Applied Biosystems 3130 3130xl Genetic Analyzers Quick Reference Card 4362825 Applied Biosystems 3130 3130xl Genetic Analyzers AB Navigator Software 4359472 Administrator Guide Applied Biosystems 3130 3100xl DNA Analyzers User Guide 4331468 Applied Biosystems 3730 3730xl Genetic Analyzer Getting Started Guide 4359476 Quantifiler Kits Quantifiler Human DNA Quantification Kit and Quantifiler 4344790 Y Human Male DNA Quantification Kit User s Manual PrepFiler Forensic DNA Extraction Kit User Guide 4390932 GeneMapper ID Software Version 3 1 Human Identification Analysis User 4338775 Guide GeneMapper ID Software Versions 3 1 and 3 2 Human Identification 4335523 Analysis Tutorial Installation Procedures and New Features for GeneMapper ID Software 4352543 v3 2 User Bulletin GeneMapper ID X Software Version 1 0 Getting Started Guide 4375574 GeneMapper ID X Software Version 1 0 Quick Reference Guide 4375670 GeneMapper ID X Software Version 1 0 Reference Guide 4375671 GeneMapper ID X Software Version 1 1 Mixture Analysis Getting Started 4396773 Guide AmpF4STR9 PCR Amplification Kit User s Guide 101 Documentation Part Document title number GeneMapper ID X Software Versio
17. Faint or no signal from both the 007 and the DNA test samples at all loci continued Degraded formamide Check the storage of formamide do not thaw and refreeze multiple times Try Hi Di Formamide AmpF4STR9 PCR Amplification Kit User s Guide 87 Appendix A Troubleshooting Observation Possible causes Recommended actions Positive signal from AmpF STR Control DNA 007 but partial or no signal from DNA test samples Quantity of test DNA sample is below assay sensitivity Quantify DNA and add 1 0 ng of DNA Repeat test Test sample contains high concentration of PCR inhibitor for example heme compounds certain dyes Quantify DNA and add minimum necessary volume Repeat test Wash the sample in a Centricon 100 centrifugal filter unit Repeat test Test sample DNA is severely degraded If possible evaluate the quality of DNA sample by running an agarose gel If DNA is degraded with an increased amount of DNA or use the AmpF STR MiniFiler Kit If possible evaluate the quality of DNA sample by running an agarose gel If DNA is degraded reamplify with an increased amount of DNA or use the AmpF STR MiniFiler Kit Redilute DNA using low TE Buffer with 0 1 mM EDTA More than two alleles present at a locus Presence of exogenous DNA Use appropriate techniques to avoid introducing foreign DNA during laboratory handling A
18. Size Standard peaks in its sizing algorithm 75 100 139 150 160 200 300 350 400 and 450 Use the following procedure to create the size standard for the AmpF STR NGM Kit 1 Select Tools GeneMapper ID X Manager to open the GeneMapper D X Manager 74 AmpF amp STR PCR Amplification Kit User s Guide Set up GeneMapper ID X Software for data analysis 2 Select the Size Standards tab then click New GeneMapper ID X Manager Find Name Containing r Y r Projects Analysis Methods Table Settings Plot Settings Matrices Size Standards Report Settings Name Last Saved Owner Type Description CE HID 65500 75 400 2007 08 09 13 23 gmidx Advanced CE_F_HID_G5500 75 450 2007 08 09 13 24 60 gmidx Advanced CE_G5_HID_GS5nn 2006 10 11 13 12 2 gmidx Advanced 3 Complete the Name field as shown below or with a name of your choosing In the Security Group field select the Security Group appropriate to your software configuration from the drop down list In the Size Standard Dye field select Orange In the Size Standard Table enter the sizes specified in Create a size standard on page 74 Size Standard Editor Edit Size Standard Description CE G5 NGM GS500 Security Group GeneMapper ID X Security Group v Description
19. Stop PE 0000 Size 1000 TBD Min Peak Half Width Smoothing None Gu Polynomial Degree Light is C Heavy Peak Window Size Baseline Window 5 pts Peak Start r Size Calling Method Peak End Smoothing and Baselining Order Least Squares Cubic Spline Interpolation Local Southern Method Global Southern Method Factory Defaults OK IMPORTANT To be determined TBD indicates values to be determined your laboratory Laboratories must perform the appropriate internal studies to determine the peak amplitude thresholds for interpretation of kit data Fields include Peak amplitude thresholds The software uses these parameters to specify the minimum peak height in order to limit the number of detected peaks Although GeneMapper JD Software displays peaks that fall below the specified amplitude in electropherograms the software does not label or determine the genotype of these peaks Size calling method NGM kit has been validated using the 3 Order Least Squares sizing method in combination with the GeneScan 500 LIZ size standard Alternative sizing methods should be selected only after extensive evaluation as part of an internal validation study in the user s laboratory 56 AmpF amp STR PCR Amplification Kit User s Guide Gen
20. User Guide Part no 4338775 CE_GS_NGM_GS300 Neither the 250 nt nor the 340 nt peak is included in the size standard definition These peaks can be used as an indicator of precision within a run 3 Click Analyze enter a name for the project in the Save Project dialog box then click OK to start analysis e e 76 The status bar displays the progress of analysis as a completion bar extending to the right with the percentage completed indicated The table displays the row of the sample currently being analyzed in green or red if analysis failed for the sample AmpF amp STR PCR Amplification Kit User s Guide For more information The Analysis Summary tab is displayed upon completion of the analysis The figure below shows the analysis summary window after analysis ID X NGM Analysis Example gmidx Is Logged In Database FOSGREENRXLO4 File Edi Analysis View Tools Admin Help iG m gt Table Setting 31XX Data Analysis v El Par S ss Project Samples Analysis Summary Genotypes 2110217 Mull Analysis Summary Select run Folder to display Sample Status Total of Samples Ld Unanalyzed 0 Analyzed 18 GF Analysis Setting Changed 0 Summary Generation Date Feb 25 2011 10 33 37 AM Click a link below to display a filtered Samples Table containing only the samples selected Allelic Ladder Quality per run folde
21. for 11 min Master Mix not vortexed thoroughly before aliquoting Vortex Master Mix thoroughly AmpF STR NGM Primer Set exposed to too much light Store Primer Set protected from light GeneAmp PCR System malfunction Refer to the thermal cycler user s manual and check instrument calibration Incorrect thermal cycler parameters Check the protocol for correct thermal cycler parameters Tubes plate not seated tightly in the thermal cycler during amplification Push reaction tubes plate firmly into contact with block after first cycle Repeat test Wrong PCR reaction tubes or plate Use Applied Biosystems MicroAmp Reaction Tubes with Caps or the MicroAmp Optical 96 Well Reaction Plate for the GeneAmp PCR System 9700 MicroAmp Base used with tray retainer set and tubes in GeneAmp PCR System 9700 Remove MicroAmp Base from tray retainer set and repeat test Insufficient PCR product electrokinetically injected For ABI PRISM 3100 Avant or Applied Biosystems 3100 3130x runs Mix 1 0 uL of PCR product and 10 of Hi Di Formamide GeneScan 500 LIZ Size Standard solution For Applied Biosystems 3500 3500xL instrument runs Mix 1 0 uL of PCR product and 10 uL of Hi Di Formamide GeneScan 500 LIZ9 Size Standard solution For ABI Prism 310 instrument runs Mix 0 75 uL of PCR product and 24 25 uL of Hi Di Formamide GeneScan 500 LIZ9 Size Standard solution
22. product name SDS part number or other information that appears in the SDS of interest Select the language of your choice then click Search 3 Find the document of interest right click the document title then select any of the following Open To view the document Print Target To print the document Save Target As To download a PDF version of the document to a destination that you choose Note For the SDSs of chemicals not distributed by Applied Biosystems contact the chemical manufacturer AmpF amp STR PCR Amplification Kit User s Guide 95 Appendix Safety Chemical waste safety Chemical waste WARNING HAZARDOUS WASTE Refer to Material Safety Data Sheets hazards and local regulations for handling and disposal WARNING CHEMICAL WASTE HAZARD Wastes produced by Applied Biosystems instruments are potentially hazardous and can cause injury illness or death WARNING CHEMICAL STORAGE HAZARD Never collect or store waste in a glass container because of the risk of breaking or shattering Reagent and waste bottles can crack and leak Each waste bottle should be secured in a low density polyethylene safety container with the cover fastened and the handles locked in the upright position Wear appropriate eyewear clothing and gloves when handling reagent and waste bottles Chemical waste minimize the hazards of chemical waste safety guidelines e Read and u
23. Contents and Storage Component Description 200X Volume 1000X Volume Storage AmpF STR NGM Contains forward and reverse 1 tube 1 0 mL 1 bottle 5 0 mL 15 to 25 C on Primer Set primers to amplify human DNA receipt 2 to 8 C targets after initial use AmpF STR NGM Master Mix Contains enzyme salts dNTPs carrier protein and 0 05 sodium azide 2 tubes 1 0 mL each 1 bottle 10 0 mL 15 to 25 C on receipt 2 to 8 C after initial use AmpF STR NGM Contains amplified alleles 1 tube 50 0 HL 1 tube 75 0 HL 15 to 25 C on Allelic Ladder See Table 1 on page 11 for a list of alleles included in the allelic ladder AmpF STR Contains 0 10 ng uL human 1 tube 0 3 mL 1 tube 0 3 mL 2 to 8 C Control DNA 007 male 007 DNA in 0 02 sodium azide and buffert See Table 1 on page 11 for profile The AmpF STR Control DNA 007 is included at a concentration appropriate to its intended use as an amplification control to provide confirmation of the capability of the kit reagents to generate a profile of expected genotype The AmpF STR Control 007 is not designed to be used as a DNA quantitation control and laboratories may expect to see variation from the labelled concentration when quantitating aliquots of the AmpF STR Control DNA 007 Standards for samples For the AmpF STR NGM Kit the panel of standards needed for PCR amplification PCR pro
24. Guide 67 Section 4 1 Data Analysis s Import Marker Stutter NGM Analysis Files GMIDX v amp NGM_bins_v2x txt NGM_panel_v2x txt My Recent NGM_stutter_v2x txt Documents My Documents Files of type all Files File name INGM_stutter_v2x txt 10 View the imported marker stutters in the navigation pane a Select the NGM_panel_v2X folder to display its list of markers in the right pane b Double click the NGM_panel_v2X folder to display its list of markers below it c Double click D2281045 to display the Stutter Ratio amp Distance view for the marker in the right pane Because 02251045 has a trinucleotide repeat unit it produces a higher level of plus stutter than tetranucleotide markers and so requires the use of a plus stutter filter The settings for the D22S1045 plus stutter filter can be seen in the table in the right pane Other markers may not require a plus stutter filter in which case the settings for plus stutter are left blank Panel Manager File Edit Bins View Help ax NNI B bins ma BEBNHBEB o E s 5 rursus ANN Please enter the stutter filter s for 02251045 marker here If left blank the global stutter filter will be applied amp C9NGM panel v2X 6 Minus Stutter Plus Stutter
25. M Anjos M J Vieira D N and Vide M C 6 5 2000 A study on ten short tandem repeat systems African immigrant and Spanish population data Forensic Sci Int 110 3 167 177 AmpFISTR amp NGM PCR Amplification Kit User s Guide Gill Fereday L Morling N Schneider 2006 New multiplexes for Europe Amendments and clarification of strategic development Forensic Sci Int 163 2006 155 157 Glock B Dauber E M Schwartz D W Mayr W R 1997 Additional variability at the D12S391 STR locus in an Austrian population sample sequencing data and allele distribution Forensic Sci Int 90 197 203 Grossman P D Bloch W Brinson E Chang C C Eggerding F A Fung S Iovannisci D M Woo S Winn Deen E S 1994 High density multiplex detection of nucleic acid sequences oligonucleotide ligation assay and sequence coded separation Nucleic Acids Res 22 4527 4534 Grubwieser P Muhlmann R Berger B Niederstatter H Palvic M Parson W 2006 A new mini STR multiplex displaying reduced amplicon lengths for the analysis of degraded DNA Int J Legal Med 120 115 120 Guo S W and Thompson E A 1992 Performing the exact test of Hardy Weinberg proportion for multiple alleles Biometrics 48 361 372 Guthmiller J M Vargas K G Srikantha R Schomberg L L Weistroffer P L McCray P B and Tack B F 2001 Susceptibilities of oral bacteria and yeast to mammalian cath
26. Mixture Analysis Tool Getting Started Guide Part no 4396773 AmpF amp STR PCR Amplification Kit User s Guide 77 O D 5 D D gt 2 0 Section 4 1 Data Analysis GeneMapper ID X Software Version 1 1 Mixture Analysis Tool Quick Reference Guide Part no 4402094 GeneMapper ID X Software Version 1 2 Quick Reference Guide Part no 4426482 78 AmpFSTR PCR Amplification Kit User s Guide Number 4466844 Rev 04 2011 Chapter 5 Experiments and Results AmpFSTR NGM PCR Amplification Kit User s Guide AmpFSTR PCR Amplification Kit User s Guide Experiments and Results Content in this chapter to come at PRC AmpF amp STR PCR Amplification Kit User s Guide 83 Chapter 5 Experiments and Results 84 AmpFSTR PCR Amplification Kit User s Guide Number 4466844 Rev 04 2011 Troubleshooting Follow the actions recommended in this appendix to troubleshoot problems that occur during analysis Observation Possible causes Recommended actions Faint or no signal from both the 007 and the DNA test samples at all loci Incorrect volume or absence of either AmpF STR NGM Master Mix or AmpF STR NGM Primer Set Repeat amplification using correct reagent volumes No activation of enzyme Repeat amplification making sure to hold reactions initially at 95
27. User s Guide Number 4466844 Rev 04 2011 Chapter 4 Data Analysis AmpFSTR NGM PCR Amplification Kit User s Guide AmpFSTR PCR Amplification Kit User s Guide Data Analysis This chapter covers GeneMapper ID 48 B Before youstart Lo PERE EDR ORR kuqa 48 Set GeneMapper ID Software for data analysis 49 m Analyze and edit sample files with GeneMapper ID Software 59 For more 61 Section 4 1 GeneMapper ID X Software 63 B Before you a HOE Ree B VES 63 Set GeneMapper ID X Software for data 64 Analyze and edit sample files with GeneMapper ID X Software 76 For more 77 AmpF amp STR PCR Amplification Kit User s Guide 47 Chapter 4 Data Analysis GeneMapper ID Software Before you start GeneMapper ID Software is an automated genotyping software for forensic casework databasing and paternity data analysis After electrophoresis the Data Collection Software stores information for each sample in an fsa file Using GeneMapper ID Software v3 2 1 software you can then analyze and interpret the data from the fSa files Note Refer to Instru
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29. extreme situations SDSs Safety Data Sheets SDSs for any chemicals supplied by Applied Biosystems or Ambion are available to you free 24 hours a day For instructions on obtaining SDSs see Obtaining SDSs on page 95 IMPORTANT For the SDSs of chemicals not distributed by Applied Biosystems or Ambion contact the chemical manufacturer AmpF amp TR NGM PCR Amplification Kit User s Guide 7 Preface 8 AmpFSTR PCR Amplification Kit User s Guide Overview This chapter covers B Product Overview si 0 10 Workflow overview 14 Instrument and software 15 Materials and 17 AmpF amp STR PCR Amplification Kit User s Guide 9 Chapter 1 Overview Product overview Purpose Product description About the primers 10 The AmpF STR NGM PCR Amplification Kit is a short tandem repeat STR multiplex assay that amplifies 14 tetranucleotide repeat loci and one trinucleotide repeat locus D22S1045 The kit simultaneously coamplifies the 10 loci contained in the AmpF STR SGM Plus kit 0351358 vWA 0165539 0251338 0851179 D21S11 018551 0195433 01 and FGA together with 2 highly polymorphic STR loci 0151656 and D12S391 3 mini STR loci 01051248 02251045 and 025441 and the gender determination locus Amelogen
30. m Set up the 3500 3500xL instrument for electrophoresis 37 Prepare samples for electrophoresis the 3500 3500xL instrument 38 Section 3 3 310 Instrument 41 Set up the 310 instrument for electrophoresis 4 Prepare samples for electrophoresis the 310 instrument 42 AmpF amp STR PCR Amplification Kit User s Guide 33 Chapter 3 Electrophoresis Allelic ladder requirements To accurately genotype samples you must run an allelic ladder sample along with the unknown samples For samples run on the Applied Biosystems 3500 Series Genetic Analyzers Run at least one allelic ladder per every set of 24 samples Applied Biosystems 3500xL One ladder per injection One injection 24 samples 23 samples 1 allelic ladder Applied Biosystems 3500 One ladder for every 3 injections One injection 8 samples ABI PRISM 3100 and Applied Biosystems 3130 Genetic Analyzers Run at least one allelic ladder per every set of 16 samples Applied Biosystems 3130xl or ABI PRISM 3100 systems One ladder injection one injection 16 samples 15 samples 1 allelic ladder Applied Biosystems 3130 or ABI PRISM 3100 Avant One ladder for every 4 injections one injection 4 samples ABI PRISM 310 Genetic Analyzer Run at least one allelic ladder for every 10 sample injections IMPORTANT Variat
31. minimum The following table lists Data Collection Software and the run modules that can be used to analyze AmpF STR NGM Kit PCR products For details on the procedures refer to the documents listed in the table Data mS Collection gp ein Run modules and conditions References Software Applied 3500 Data Windows HID36_POP4 Applied Biosystems Biosystems Collection XP Injection conditions 1 2kV 15 sec 3900 3500xL Genetic Analyzer 3500 Software User Guide Part no 4401661 v1 0 or Dye Set G5 2 3500 and 3500xL Genetic Applied Windows HID36_POP4 Analyzers Quick Reference PET vista Injection conditions 1 2kV 24 sec Part no 4401662 Dye Set G5 AmpF amp STR PCR Amplification Kit User s Guide 37 a 5 e 5 2 Section 3 2 Electrophoresis Prepare samples for electrophoresis on the 3500 3500xL instrument Prepare the samples for capillary electrophoresis on the 3500 3500xL instrument immediately before loading 1 38 Calculate the volume of Hi Di Formamide and GeneScan 500 117 Size Standard needed to prepare the samples using the table below Reagent Volume per reaction pL GeneScan 500 LIZ Size Standard 0 5 uL Hi Di Formamide 9 5 uL Note Include additional samples in your calculations to provide excess volume for the loss that occurs during reagent transfers IMPORTANT The volume of s
32. or virtual bin Note If a sample allele peak 15 called as an off ladder allele verify the sample result according to your laboratory s protocol If you are using the GeneMapper D X Software to perform Human Identification HID analysis with AmpF STR kits go to Set up GeneMapper ID X Software for data analysis on page 64 or refer to the GeneMapper ID X Software Version 1 0 Human Identification Analysis Getting Started Guide Part no 4375574 AmpF amp STR PCR Amplification Kit User s Guide 48 O 5 0 0 e E GeneMapper ID Software Setup Workflow GeneMapper ID To analyze sample fsa files using GeneMapper ID Software v3 2 1 for the first Software for data analysis Import panels and bins into the Panel Manager as explained in Import panels and bins on page 49 Create an analysis method as explained in Create a HID analysis method on page 52 Create a size standard as explained in Create a HID size standard on page 58 Define custom views of analysis tables Refer to the GeneMapper ID Software Versions 3 1 and 3 2 Human Identification Analysis Tutorial Part no 4335523 for more information Define custom views of plots Refer to the GeneMapper ID Software Versions 3 1 and 3 2 Human Identification Analysis Tutorial Part no 4335523 for more information Import panels and bi
33. select the Security Group appropriate to your software configuration from the drop down list The Description and Instrument fields are optional 54 AmpF4 TR PCR Amplification Kit User s Guide GeneMapper ID Software Allele tab settings Analysis Method Editor HID General Allele Detector Peak Quality Quality Flags Bin Use marker specific stutter ratio available Plus Stutter Distance From To Marker Repeat Type Tri Tetra Cut off hn pn pn pn S Minusa Ratio bo bo bo po lt Distance From hn hn foo 5 joo pn pn pn Minus Stutter Ratio bo foo foo ho 5 Minus Stutter Distance From 225 25 pn CD to Ez foo F Plus Stutter Ratio ha hn pn pn pz po ho Amelogenin Cutoff Range Filter Factory Defaults OK Cancel In the Bin Set field select the NGM_bins_v2 bin set imported previously and configure the stutter distance parameters as shown GeneMapper ID Software v3 2 1 allows you to specify four types of marker repeat motifs tri tetra penta and hexa You can enter parameter values for each type of repeat in the appropriate column The Use marker specific stutter ratio if available check box is selected by default Consequently the software applies the stutter ratio filters supplied in the NGM_panel_v2 file Gen
34. 000 Analyze and edit Analyze a project sample files with GeneMapper D 1 In the Project window select File Add Samples to Project then navigate to Software the disk or directory containing the sample files AmpF4STR9 PCR Amplification Kit User s Guide 59 Chapter 4 Data Analysis 2 Apply analysis settings to the samples in the project The names of the settings shown are the names suggested in the sections above If you named the settings differently select the name you specified Parameter Settings Sample Type Select the sample type Analysis Method NGM_AnalysisMethod_v2 Panel NGM_panel_v2 Size Standard CE G5 NGM GS500 Size Standard For more information about how the Size Caller works refer to the ABI Prism GeneScan Analysis Software for the Windows NT Operating System Overview of the Analysis Parameters and Size Caller User Bulletin Part no 4335617 CE G5 05500 size standard fragments defined in the AmpF STR NGM Kit 75 100 139 150 160 200 300 350 400 and 450 For additional information about size standards refer to the GeneMapper ID Software Version 3 1 Human Identification Analysis User Guide Part no 4338775 65 05500 Neither the 250 nt nor 340 nt peak is included in the size standard definition These peaks can be used as an indicator of precision within a run 3 Click Analyze enter a name for the pr
35. 101 How to obtain support 102 Bibliography 3243 29 XXy 103 1 109 amp PCR Amplification Kit User s Guide Preface Safety information Note For general safety information see this Preface and Appendix C Safety on page 93 When a hazard symbol and hazard type appear by an instrument hazard see the Safety Appendix for the complete alert For all chemicals read the SDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves Safety alert Four safety alert words appear in Applied Biosystems user documentation at points words the document where you need to be aware of relevant hazards Each alert word IMPORTANT CAUTION WARNING DANGER implies a particular level of observation or action as defined below IMPORTANT Indicates information that is necessary for proper instrument operation accurate chemistry kit use or safe use of a chemical CAUTION Indicates a potentially hazardous situation that if not avoided may result in minor or moderate injury It may also be used to alert against unsafe practices WARNING Indicates a potentially hazardous situation that if not avoided could result in death or serious injury DANGER Indicates an imminently hazardous situation that if not avoided will result in death or serious injury This signal word is to be limited to the most
36. 7 4 bmbl od nih gov Occupational Safety and Health Standards Bloodborne Pathogens 29 CFR 1910 1030 www access gpo gov nara cfr waisidx_01 29cfr1910a_01 html Your company s institution s Biosafety Program protocols for working with handling potentially infectious materials Additional information about biohazard guidelines is available at www cdc gov 98 AmpF amp STR PCR Amplification Kit User s Guide Chemical alerts Chemical alerts For the definitions of the alert words IMPORTANT CAUTION WARNING and DANGER see Safety alert words on page 7 General alerts for Avoid contact with skin eyes and or clothing Read the SDS and follow the handling all chemicals instructions Wear appropriate protective eyewear clothing and gloves Specific CAUTION CHEMICAL HAZARD AmpFLSTR NGM PCR chemical alerts Amplification Kit may cause eye skin and respiratory tract irritation Sodium azide may react with lead and copper plumbing to form highly explosive metal azides WARNING CHEMICAL HAZARD POP 4 Polymer for 3130 3130 Genetic Analyzers causes skin eye and respiratory tract irritation WARNING CHEMICAL HAZARD Running Buffer 10 causes skin eye and respiratory tract irritation WARNING CHEMICAL HAZARD Hi Di Formamide is harmful if swallowed inhaled or absorbed through skin and causes irritation to skin eyes and respiratory tract It affects the central nervous sys
37. GM_v2X 51051248 Blue 720 1270 1215 4 8 9 10 11 12 13 14 15 16 17 18 VIA Blue 149 0 214 3 1416 4 11 12 13 14 15 16 17 18 19 20 21 22 2 7 0165539 Blue 223 6 277 6 9 10 4 none 5 8 9 10 11 12 13 14 15 0165539 0251338 Blue 281 6 356 0 20 23 4 15 16 17 18 19 20 21 22 23 24 25 26 27 0251338 AMEL Green 100 0 108 0 9 aY AMEL D851179 Green 117 9 174 9 12 13 4 8 9 10 11 12 13 14 15 16 17 18 19 0851179 TF J n FJ 021511 Green 178 8 2498 28 31 4 24 24 2 25 26 27 28 28 2 29 29 2 30 30 018551 018551 Green 259 5 347 5 12 15 4 7 9 10 10 2 11 12 13 13 2 14 14 2 15 11 02251045 02251045 Yelow 260 1200 11 16 3 none 8 9 10 11 12 13 14 15 16 17 18 19 0195433 D195433 Yellow 122 3 166 3 14 15 4 9 10 11 12 12 2 13 13 2 14 14 2 15 15 01 hn A EL T Q U FEA Yelow 176 4 2211 79 3 4 4 5 6 7 8 9 9 3 10 11 13 3 D25441 FGA Yellow 221 6 372 0 24 26 4 none 17 18 19 20 21 22 23 24 25 26 26 2 27 0351358 D25441 Red 745 134 1415 4 9 10 11 11 3 12 13 14 15 16 0151656 D351358 Red 114 4 168 4 15 16 4 12 13 14 15 16 17 18 19 0125391 n Y O qI A U iN An Padi dE D151656 Red 170 0 2240 13 16 4 none 9 10 11 12 13 14 14 3 15 15 3 16 16 3 D125391 Red 225 0 287 0 18 19 4 14 15 16 17 18 19 19 3 20 21 22 23 24 as Reference Samples
38. Guide Part no 4335393 3130 3130x Analyzer materials 96 Well Plate Septa 4315933 Reservoir Septa 4315932 3100 3130x Genetic Analyzer Capillary Array 36 cm 4315931 POP 4 Polymer for 3130 3130 Genetic Analyzers 4352755 3130 3130 Genetic Analyzer Autosampler Plate Kit 96 well 4316471 GeneScan 500 LIZ Size Standard 4322682 Running Buffer 10x 402824 DS 33 Matrix Standard Kit Dye Set G5 4345833 MicroAmp Optical 96 Well Reaction Plate N8010560 For a complete list of parts and accessories for the 3130 instrument refer to Appendix A of the Applied Biosystems 3130 3130xl Genetic Analyzers Maintenance Troubleshooting and Reference Guide Part no 4352716 310 Analyzer materials 310 DNA Analyzer Capillary Array 47 cm 402839 0 5 mL Sample Tray 5572 96 Well Tray Adaptor for 9700 thermal cycler trays 4305051 GeneScan 500 LIZ Size Standard 4322682 Running Buffer 10X 4335643 Genetic Analyzer Septa Retainer Clips for 96 Tube Sample Tray 402866 Genetic Analysis Sample Tubes 0 5 mL 401957 Septa for 0 5 mL Sample Tubes 401956 DS 33 Matrix Standard Set 6 FAM VIC NED and LIZ dyes for 4318159 ABI PRISM 310 377 systems MicroAmp 8 Tube Strip 0 2 mL N8010580 MicroAmp 96 Well Base holds 0 2 mL reaction tubes N8010531 90 AmpF4STR9 PCR Amplification Kit User s Guide Materials and equipment not included
39. Loci amplified by the kit Product overview The following table shows the loci amplified their chromosomal locations and the corresponding fluorescent marker dyes The AmpF STR NGM Allelic Ladder is used to genotype the analyzed samples The alleles contained in the allelic ladder and the genotype of the AmpF STR Control DNA 007 are also listed in the table Table 1 AmpF STR NGM Kit loci and alleles Chromosome Alleles included in AmpF STRS NGM Dye Control DNA Locus designation location Kit Allelic Ladder label 007 D1081248 10q26 3 8 9 10 11 12 13 14 15 16 17 18 6 FAM 12 15 vWA 12p13 31 11 12 13 14 15 16 17 18 19 20 21 22 6 FAM 14 16 23 24 016 539 16q24 1 5 8 9 10 11 12 13 14 15 6 FAM 9 10 02 1338 2435 15 16 17 18 19 20 21 22 23 24 25 6 FAM 20 23 26 27 28 Amelogenin p22 1 22 3 X Y vic X Y Y p11 2 D8S1179 8q24 13 8 9 10 11 12 13 14 15 16 17 18 19 VIC 12 13 021 11 21q11 2 q21 24 24 2 25 26 27 28 28 2 29 29 2 30 VIC 28 31 30 2 31 31 2 32 32 2 33 33 2 34 34 2 35 35 2 36 37 38 D18S51 18q21 33 7 9 10 10 2 11 12 13 13 2 14 14 2 15 12 15 16 17 18 19 20 21 22 23 24 25 26 27 D2281045 22q12 3 8 9 10 11 12 13 14 15 16 17 18 19 NED 11 16 D19S433 19q12 9 10 11 12 12 2 13 13 2 14 14 2 15 NED 14 15 15 2 16
40. M master mix volume per reaction 25 materials and equipment included in kit 17 not included in kit 89 multicomponent analysis 15 16 N negative control sample preparation 25 O operating systems 15 35 37 41 performing 26 setup tools 22 thermal cycling conditions programming 26 work area setup 22 work areas 22 110 positive control sample preparation 25 primers volume per reaction 25 Q quantification DNA 23 R radioactive waste handling 97 reaction mix for PCR 25 reagents user supplied 23 run module electrophoresis 35 37 41 safety biological hazards 98 chemical waste 96 guidelines 94 96 sample files fsa 49 64 sample preparation 25 DNA negative control 25 DNA positive control 25 standards 17 SDSs about 7 description 94 obtaining 95 102 setup tools PCR 22 software instrument compatibility 15 thermal cycling programming conditions 26 training information on 102 U user supplied reagents 23 W WARNING description 7 waste disposal guidelines 96 waste profiles description 96 work area amplified DNA tools 22 PCR tools 22 setup 22 workflow overview 14 PCR Amplification Kit User s Guide 299 Headquarters 5791 Van Allen Way Carlsbad 92008 USA Phone 1 760 603 7200 Toll Free in USA 800 955 6288 For support visit www appliedbiosystems com support technologies www lifetechnologies
41. USER GUIDE applied biosystems by Life technologies AmpF STR NGM PCR Amplification Kit Publication Part Number 4466844 Rev Revision Date April 2011 technologies AmpF STR PCR Amplification Kit User s Guide KS PP Copyright 2011 Life Technologies Corporation All rights reserved For Research Forensic or Paternity Use Only Not intended for any animal or human therapeutic or diagnostic use Information in this document is subject to change without notice Applied Biosystems assumes no responsibility for any errors that may appear in this document APPLIED BIOSYSTEMS DISCLAIMS ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT EXPRESSED OR IMPLIED INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE TO THE FULLEST EXTENT ALLOWED BY LAW IN NO EVENT SHALL APPLIED BIOSYSTEMS BE LIABLE WHETHER IN CONTRACT TORT WARRANTY OR UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPECIAL INCIDENTAL INDIRECT PUNITIVE MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT INCLUDING BUT NOT LIMITED TO THE USE THEREOF WHETHER OR NOT FORESEEABLE AND WHETHER OR NOT APPLIED BIOSYSTEMS IS ADVISED OF THE POSSIBILITY OF SUCH DAMAGES NOTICE TO PURCHASER Use of the AmpF STR NGM PCR Amplification Kit is covered by US patent claims and patent claims outside the US The purchase of this product includes a limited non transferable immunity from suit under
42. ab fsc current standards 2004 03 standards02 htm Rousset 2008 Genepop 007 A complete reimplementation of the Genepop software for Windows and Linux Molecular Ecology Resources 8 103 106 AmpFISTR amp NGM PCR Amplification Kit User s Guide Sensabaugh 1982 Biochemical markers of individuality In Saferstein R ed Forensic Science Handbook Prentice Hall Inc New York pp 338 415 Sharma V and Litt M 1992 Tetranucleotide repeat polymorphism at the D21S11 locus Hum Mol Genet 1 67 Shin C H Jang P Hong K M Paik 2004 Allele frequencies of 10 STR loci in Koreans Forensic Sci Int 140 133 135 Smith R N 1995 Accurate size comparison of short tandem repeat alleles amplified by PCR Biotechniques 18 122 128 Sparkes R Kimpton C Watson S Oldroyd N Clayton T Barnett L Arnold J Thompson C Hale R Chapman J Urquhart A and Gill P 1996a The validation of a 7 locus multiplex STR test for use in forensic casework I Mixtures ageing degradation and species studies nt Legal Med 109 186 194 Sparkes R Kimpton C Gilbard S Carne P Andersen J Oldroyd N Thomas D Urquhart A and Gill 1996b The validation of a 7 locus multiplex STR test for use in forensic casework Artifacts casework studies and success rates Int J Legal Med 109 195 204 Straub R E Speer M C Luo Y Rojas K Overhauser J Ott J and Gilli
43. ak height thresholds maximum peak height threshold and the minimum peak height ratio threshold for reliable interpretation of AmpF STR NGM Kit data AmpF4STR9 PCR Amplification Kit User s Guide 73 O 5 D gt 2 0 Section 4 1 Data Analysis SQ amp GQ tab settings Analysis Method Editor General Allele Peak Detector Peak Quality 50 amp GQ Settings Quality weights are between 0 and 1 r Sample and Control GQ Weighting Broad Peak BD Allele Number AN Out of Bin Allele BIN Low Peak Height LPH Overlap OVL Max Peak Height MPH Marker Spike SPK Off scale OS Peak Height Ratio PHR Control Concordance CC Weight 1 0 Only applicable to controls 59 Weighting Broad Peak BD Spike 55 5 Off scale 05 Sizing Quality From 0 75 to1 0 From0 0to 0 25 Genotype Quality From 0 75 to1 0 From0 0to 0 25 Reset Defaults IMPORTANT The values shown are the software defaults and are the values used by Applied Biosystems during developmental validation Laboratories must perform appropriate internal validation studies to determine the appropriate values to use 4 Click Save Create size size standard for the AmpF STR NGM PCR Amplification Kit uses the standard following GeneScan 500 LIZ
44. am T C 1993 A microsatellite genetic linkage map of human chromosome 18 Genomics 15 48 56 Straub R E Speer M C Luo Y Rojas K Overhauser J Ott J and Gilliam T C 1993 A microsatellite genetic linkage map of human chromosome 18 Genomics 15 48 56 Suido H Nakamura Mashimo PA Zambon J J and Genco R J 1986 Arylaminopeptidase activities of the oral bacteria Dent Res 65 1335 1340 Szibor R Lautsch S Plate I Bender K Krause D 1998 Population genetic data of the STR HumD3S1358 two regions of Germany Int J Legal Med 111 3 160 1 Waiyawuth W Zhang L Rittner C Schneider 1998 Genetic analysis of the short tandem repeat system D12S391 in the German and three Asian populations Forensic Sci Int 94 25 31 Wallin J M Buoncristiani M R Lazaruk K D Fildes N Holt C L Walsh 1998 SWGDAM validation of the AmpF STR blue PCR amplification kit for forensic casework analysis J Forensic Sci 43 854 870 Wallin J M Holt C L Lazaruk K D Nguyen T H Walsh P S 2002 Constructing universal multiplex PCR systems for comparative genotyping J Forensic Sci 47 52 65 Walsh PS Fildes N J Reynolds 1996 Sequence analysis and characterization of stutter products at the tetranucleotide repeat locus vWA Nucleic Acids Res 24 2807 2812 AmpFISTR NGM Amplification Kit User s Guide 107 Bibliography 108 Watson S
45. apper Manager AmpF amp STR PCR Amplification Kit User s Guide GeneMapper ID Software Manager Projects Analysis Methods Table Settings Plot Settings Matrices Size Standards ILast Saved Owner Instrument Analysis Type Descrip HID_Advanced 2009 06 18 16 22 2 gmid HID HID_Classic 2007 08 06 10 03 0 gmid HID Microsstelite Default 2004 05 28 11 34 3 gmid Microsatellite Factor 2 Select the Analysis Methods tab then click New to open the New Analysis Method dialog box 5 lt 9 to 122 E 3 Select HID and click OK to open Analysis Method Editor with the General Tab selected 4 The figures below show the settings for each tab of the Analysis Method Editor Configure settings as shown unless the instructions state otherwise Note The Analysis Method Editor closes when you save your settings See step 5 on page 58 To complete this step quickly do not save the analysis method until you finish entering settings in all of the tabs AmpF amp STR PCR Amplification Kit User s Guide 53 Chapter 4 Data Analysis General tab settings Analysis Method Editor HID In the Name field either type the name as shown for consistency with files supplied with other AmpF STR kits or enter a name of your choosing In the Security Group field
46. ar appropriate personal protective equipment when handling chemicals for example safety glasses gloves or protective clothing For additional safety guidelines consult the SDS Minimize the inhalation of chemicals Do not leave chemical containers open Use only with adequate ventilation for example fume hood For additional safety guidelines consult the SDS Check regularly for chemical leaks or spills If a leak or spill occurs follow the manufacturer s cleanup procedures as recommended in the SDS Comply with all local state provincial or national laws and regulations related to chemical storage handling and disposal About SDSs Chemical manufacturers supply current Safety Data Sheets SDSs with shipments of hazardous chemicals to new customers They also provide SDSs with the first shipment of a hazardous chemical to a customer after an SDS has been updated SDSs provide the safety information you need to store handle transport and dispose of the chemicals safely Each time you receive a new SDS packaged with a hazardous chemical be sure to replace the appropriate SDS in your files 94 AmpF amp TR NGM PCR Amplification Kit User s Guide Chemical safety Obtaining SDS for any chemical supplied by Applied Biosystems is available to you free SDSs 24 hours day To obtain SDSs 1 Go to www appliedbiosystems com click Support then select SDS 2 Inthe Keyword Search field enter the chemical name
47. are samples for electrophoresis on the 310 instrument _ 42 AmpF4STR9 PCR Amplification Kit User s Guide 5 Contents Chapter 4 Chapter 5 Appendix A Appendix B Appendix C Data Analysis 47 GeneMapper ID Software 48 For more information 61 Section 4 1 GeneMapper D X Software 63 Before you Stari oss eei dut ee eR Edo Rede gt 63 Set up GeneMapper ID X Software for data analysis 64 Analyze and edit sample files with GeneMapper D X Software 76 For more informatlon 222 yes qaya vss aa eta es 77 Experiments and Results 83 Troubleshooting 3339 ESAE 87 Ordering Information 89 Materials and equipment included 89 dem 93 hCG 94 Chemical waste safety uuu ss ti a eee 96 Biological hazard safety 98 Chemical alerts sev tana ata ties erste ea 99 Documentation 101 Related documentation nes
48. ation 25 test sample 25 tools 22 documentation related 101 AmpF amp STR PCR Amplification Kit User s Guide E electrophoresis Data Collection Software 35 37 41 preparing samples on the 310 instrument 42 preparing samples on the 3100 3100 Avant or 3130 3130 1 instrument 36 preparing samples on the 3500 3500xL instrument 38 reagents and parts 35 37 41 references 35 37 41 run module 35 37 41 setup 35 37 41 emission spectra 16 equipment not included in kit 89 F fluorescent dyes 15 FSA sample files 49 64 FTA cards amplification 27 bloodstained 27 G GeneMapper ID Software data analysis 49 overview 15 GeneMapper ID X Software data analysis 64 overview 15 GeneScan size standard about 17 dye label 15 volume per reaction 36 38 42 guidelines chemical safety 94 chemical waste disposal 96 chemical waste safety 96 H hazards See safety Hi Di formamide volume per reaction 36 38 42 109 Index instrumentation 310 genetic analyzer 15 34 41 3100 3100 Avant genetic analyzer 15 34 35 3130 3130 genetic analyzer 15 34 35 3500 3500xL genetic analyzer 37 software compatibility 15 kit allelic ladder 17 amplification 10 contents 17 control DNA 17 description 10 fluorescent dyes 15 loci amplification 11 master mix 17 primers 10 17 24 purpose 10 reagents 17 supported instruments 10 L LIZ size standard about 17 volume per reaction 36 38 42 low TE buffer 23
49. bs of the Project window to examine the data These procedures start with the Samples tab of the Project window assuming the analysis is complete For more information For details about GeneMapper JD Software features allele filters peak detection algorithms and project editing refer to GeneMapper ID Software Versions 3 1 and 3 2 Human Identification Analysis Tutorial Part no 4335523 GeneMapper ID Software Version 3 1 Human Identification Analysis User Guide Part no 4338775 Installation Procedures and New Features for GeneMapper ID Software Version v3 2 User Bulletin Part no 4352543 AmpF amp STR PCR Amplification Kit User s Guide 61 Chapter 4 Data Analysis 62 AmpFSTR PCR Amplification Kit User s Guide Chapter 4 Data Analysis Section 4 1 GeneMapper D X Software Before you start GeneMapper D X Software is an automated genotyping software for forensic casework databasing and paternity data analysis After electrophoresis the Data Collection Software stores information for each sample in an fsa file for 310 and 31 CE instruments or an hid file for 3500 and 3500xL instruments Files in fsa format can be analyzed by any version of GeneMapper D X software that is v1 0 or higher hid files can only be analyzed by GeneMapper D X v1 2 or higher Note Refer to Instrument and software overview on page 15 for a list of compatible instruments When usin
50. ction Kit v Quantify DNA Quantifiler Duo DNA Quantification Kit v Perform PCR Amp F STR NGM PCR Amplification Kit GeneAmp PCR System 9700 Thermal Cycler Amplification should be performed on silver or gold plated silver 96 well blocks only Perform Electrophoresis 25 ABI PRISM ABI PRISM Applied Biosystems Applied Biosystems 310 Genetic 3100 3100 Avant 3130 3130xl 3500 3500xL Analyzer Genetic Analyzer Genetic Analyzer Genetic Analyzer for Human Identification Analyze Data 14 AmpF amp STR PCR Amplification Kit User s Guide Instrument and software overview Instrument and software overview This section provides information about the Data Collection Software versions required to run the AmpF STR NGM PCR Amplification Kit on specific instruments Data Collection and GeneMapper or 0 Software The Data Collection Software provides instructions to firmware running on the instrument and displays instrument status and raw data in real time As the instrument measures sample fluorescence with its detection system the Data Collection Software collects the data and stores it The Data Collection Software stores information about each sample in a sample file fsa which is then analyzed by the GeneMapper JD or ID X Software Instrument and Table2 Software specific to each instrument
51. cture and length of the tandem repeat Am J Hum Genet 62 1408 1415 Brinkman B Moller and Wiegand P 1995 Structure of new mutations in 2 STR systems ntl J Legal Med 107 201 203 Butler J M 2005 Forensic DNA Typing Burlington MA Elsevier Academic Press Butler J M Shen Y McCord B R 2003 The development of reduced size STR amplicons as tools for analysis of degraded DNA J Forensic Sci 48 1054 1064 AmpFISTR NGM PCR Amplification Kit User s Guide 103 Bibliography 104 Chakraborty R Kimmel M Stivers D Davison L and Deka R 1997 Relative mutation rates at di tri and tetranucleotide microsatellite loci Proc Natl Acad Sci USA 94 1041 1046 Chakraborty R Stivers D and Zhong Y 1996 Estimation of mutation rates from parentage exclusion data applications to STR and VNTR loci Mutat Res 354 41 48 Chakraborty and Stivers D N 1996 Paternity exclusion by DNA markers effects of paternal mutations J Forensic Sci 41 671 677 Chung D T Drabek J Opel K L Butler J M and McCord 2004 study of the effects of degradation and template concentration on the amplification efficiency of the Miniplex primer sets Forensic Sci 49 733 740 Clark J M 1988 Novel non templated nucleotide addition reactions catalyzed by procaryotic and eucaryotic DNA polymerases Nucleic Acids Res 16 9677 9686 Coble M D and Butler J M 2005 Characterizat
52. duct sizing and genotyping are Control DNA 007 A positive control for evaluating the efficiency of the amplification step and STR genotyping using the AmpF STR NGM Allelic Ladder 500 LIZ Size Standard Standard used for obtaining sizing results It contains 16 single stranded labeled fragments of 35 50 75 100 139 150 160 200 250 300 340 350 400 450 490 and 500 nucleotides This standard which has been evaluated as an internal size standard yields precise sizing results for AmpF STR Kit PCR products Order the GeneScan 500 LIZ Size Standard Part no 4322682 separately AmpF4STR9 PCR Amplification Kit User s Guide 17 Chapter 1 Overview AmpF STR NGM Allelic Ladder Allelic ladder developed by Applied Biosystems for accurate characterization of the alleles amplified by the AmpFSTRY NGM Kit The AmpF STR NGM Allelic Ladder contains most of the alleles reported for the 15 autosomal loci Refer to Table 1 on page 11 for a list of the alleles included in the AmpF STR Allelic Ladder 18 PCR Amplification Kit User s Guide Chapter 2 PCR Amplification AmpFSTR NGM PCR Amplification Kit User s Guide AmpFSTR PCR Amplification Kit User s Guide PCR Amplification This chapter covers PCR Work areas a Ra OM qa eee 22 Required user supplied materials and
53. e gt Updates amp Patches and download the file NGM Analysis Files GMIDX b Unzip the file Start the GeneMapper D X Software then log in with the appropriate user name and password IMPORTANT For logon instructions refer to the GeneMapper ID X Software Version 1 0 Getting Started Guide Part no 4375574 Select Tools Panel Manager Find then open the folder containing the panels bins and marker stutter a Select Panel Manager in the navigation pane Panel Manager File Edit Bins View Help ENS LJ Highlight this A b Select File Import Panels to open Import Panels dialog box AmpF amp STR PCR Amplification Kit User s Guide Set GeneMapper ID X Software for data analysis c Navigate to then open the NGM Analysis Files GMIDX folder that you unzipped in step 1 on page 64 5 Select NGM_panel_v2X then click Import Note Importing this file creates a new folder in the navigation pane of the Panel Manager AmpFLSTR_NGM_v2X This folder contains the panel and associated markers Import Panels in NGM Analysis Files GMIDX d c NGM bins v2X txt My Recent NGM_stutter_v2x txt Documents 2 My Documents File name NGM panel v2X txt wa Files of type Files 6 Import NGM_bins_v2X a Select the AmpFLSTR NGM v2X folder in the navigation pane
54. eMapper ID Software Peak Quality tab settings Analysis Method Editor HID General Allele Peak Detector Peak Quality Quality Flags Signal level Homozygous peak height TBD Heterozygous min peak height TBD rHeterozygote balance Q Min peak height ratio 2 morphology 5 Max peak width basepairs hs m Pull up peak E Pull up ratio 0 05 5 Allele number Max expected alleles Factory Defaults IMPORTANT be determined TBD indicates values to be determined in your laboratory Laboratories need to perform the appropriate internal validation studies to determine the minimum heterozygous and homozygous minimum peak height thresholds and the minimum peak height ratio threshold that allow for reliable interpretation of AmpFA STR NGM Kit data AmpF amp STR PCR Amplification Kit User s Guide 57 Chapter 4 Data Analysis Quality Flags tab settings Analysis Method Editor HID IMPORTANT The values shown are the software defaults and are the values used by Applied Biosystems during developmental validation Laboratories must perform appropriate internal validation studies to determine the appropriate values to use 5 Click Save Create a HID size standard The size standard for the AmpF STR NGM PCR Amplification Kit uses the following GeneScan 500 LIZ Size Standard peaks in its s
55. eMapper ID Software v3 2 1 specifies locus specific filter ratios for minus stutters but not for plus stutters in the panel file However validation studies with the NGM kit show that the trinucleotide repeat 02251045 locus produces a relatively large amount of plus stutter compared to tetranucleotide repeat loci The relatively large amount of stutter may cause the stutter peak to be labeled during routine analysis The plus stutter at the 02251045 locus can be filtered by assigning a global plus stutter filter for trinucleotide repeat loci in the Analysis Parameter file Because 02251045 is the only trinucleotide repeat locus in the NGM kit this stutter filter setting is applied only to plus stutter peaks at the D22S1045 locus The settings shown above resulted in little or no labeling of D22S1045 plus stutter peaks during validation studies at Applied Biosystems However we recommend that users determine the settings appropriate for use in their laboratory during internal validation studies AmpF amp STR PCR Amplification Kit User s Guide 55 Chapter 4 Data Analysis Peak Detector tab settings Analysis Method Editor HID EN xj General Allele Peak Detector Peak Quality Quality Flags Peak Detection Algorithm Advanced FRanges Detection Analysis Sizing Peak Amplitude Thresholds rar Range m Sizes x TBD TBD Start Pt Start Size uc TBD 6
56. ed by Applied Biosystems validation studies However it is recommended that each laboratory determine the optimum cycle number based on internal validation studies In the example shown in Figure 4 a 1 2 mm disc of a bloodstained FTA card was purified using three washes with FTA Purification Reagent and two washes with 1X low TE buffer The punch was then amplified directly in the MicroAmp tube for 24 cycles 150 130 230 270 310 350 Figure 4 AmpF STR NGM PCR Amplification Kit results from a 1 2 mm FTA bloodstain disc 24 cycle amplification analyzed on the Applied Biosystems 3130x Genetic Analyzer AmpF amp STR PCR Amplification Kit User s Guide 27 Chapter 2 Amplification 28 AmpFSTR PCR Amplification Kit User s Guide Number 4466844 Rev 04 2011 Chapter 3 Electrophoresis AmpFSTR NGM PCR Amplification Kit User s Guide AmpFSTR PCR Amplification Kit User s Guide Electrophoresis This chapter covers Allelic ladder 34 Section 3 1 3100 3100 Avant 3130 3130 instruments 35 Set up the 3100 3100 Avant or 3130 3130 1 instrument for electrophoresis 35 Prepare samples for electrophoresis on the 3100 3100 Avant 3130 3130 1 TASTUME dass nene 36 Section 3 2 3500 3500xL Series instruments 37
57. elicidins Antimicrob Agents Chemother 45 3216 3219 Hammond H Jin L Zhong Y Caskey C and Chakraborty R 1994 Evaluation of 13 short tandem repeat loci for use in personal identification applications Am J Hum Genet 55 175 189 Holt C Stauffer C Wallin J et al 2000 Practical applications of genotypic Surveys for forensic STR testing Forensic Sci Int 112 91 109 Kalinowski S T 2006 HW QuickCheck an easy to use computer program for checking genotypes for agreement with Hardy Weinberg expectations Molecular Ecology Notes 6 974 979 Kimpton C Walton A and Gill P 1992 A further tetranucleotide repeat polymorphism in the vWF gene Hum Mol Genet 1 287 Kong X Murphy K Raj T He C White P S Matise T C 2004 A combined linkage physical map of the human genome Am Hum Genet 75 1143 1148 Lareu M V Barral S Salas A Pestoni C and Carracedo A 1998 Sequence variation of a hypervariable short tandem repeat at the D1S1656 locus Int J Legal Med 111 5 244 247 Lareu M V Pestoni M C Barros F Salas A Carracedo A 1996 Sequence variation of a hypervariable short tandem repeat at the D12S391 locus Gene 182 151 153 Lazaruk K Walsh PS Oaks Gilbert D Rosenblum B B Menchen S Scheibler D Wenz H M Holt C Wallin J 1998 Genotyping of forensic short tandem repeat STR systems based on sizing precision in a capillary electrophor
58. en not in use Limit aerosol dispersal by handling sample tubes and reagents carefully Note These laboratory design resources and guidances constitute only a sample of the precautions that need to be observed when using PCR technology Refer to your laboratory s internal policies and procedures for additional information and references IMPORTANT These items should never leave the PCR setup work area Calculator Gloves disposable Marker pen permanent Microcentrifuge Microcentrifuge tubes 1 5 mL 2 0 mL or other appropriate clean tube for Master Mix preparation Microcentrifuge tube rack Pipette tips sterile disposable hydrophobic filter plugged Pipettors Tube decapper autoclavable Vortex Amplified DNA following GeneAmp PCR systems should be placed in the amplified DNA work area tools work area 22 Silver block 96 Well GeneAmp PCR System 9700 Gold plated Silver block 96 Well GeneAmp PCR System 9700 AmpF amp TR NGM PCR Amplification Kit User s Guide Required user supplied materials and reagents Required user supplied materials and reagents Kit contents and The AmpF STR NGM PCR Amplification Kit is available as either a storage 200 reaction kit or 1000 reaction kit The number of reactions is based on a 25 reaction volume See Kit contents and storage on page 17 for details on kit contents User supplied In addition to the A
59. erman population Forensic Sci Int 95 173 178 Moretti T Baumstark A Defenbaugh D Keys K Smerick J Budowle 2001 Validation of short tandem repeats STRs for forensic usage Performance testing of fluorescent multiplex STR systems and analysis of authentic and simulated forensic samples J Forensic Sci 46 3 647 660 Mulero J J Chang C W and Hennessy L K 2006 Characterization of N 3 stutter product in the trinucleotide repeat locus DYS392 J Forensic Sci 51 826 830 Nakahori Y Takenaka O and Nakagome Y 1991 A human X Y homologous region encodes amelogenin Genomics 9 264 269 National Institute of Justice Office of Law Enforcement Standards 1998 Forensic Laboratories Handbook for Facility Planning Design Construction and Moving Washington DC National Institute of Justice 76 pp Puers Hammond HA Jin Caskey CT Schumm JW Identification of repeat sequence heterogeneity at the polymorphic short tandem repeat locus HUMTHOI AATG n and reassignment of alleles in population analysis by using locus specific allelic ladder 1 Am Hum Genet 1993 Oct 53 4 953 8 Raymond M amp Rousset F 1995 GENEPOP version 1 2 population genetics software for exact tests and ecumenicism J Heredity 86 248 249 Revised Validation Guidelines Scientific Working Group on DNA Analysis Methods SWGDAM Forensic Sci Communications July 2004 Volume 6 3 Available at www fbi gov hg l
60. esis instrument Electrophoresis 19 86 93 AmpFISTR NGM PCR Amplification Kit User s Guide 105 Bibliography 106 Li H Schmidt L Wei M H Hustad Leman M I Zbar and K 1993 Three tetranucleotide polymorphisms for 10 1 0381352 0351358 0351359 Hum Mol Genet 2 1327 Magnuson V L Ally D S Nylund S J Karanjawala Z E Rayman J B Knapp J L Lowe A L Ghosh S Collins ES 1996 Substrate nucleotide determined non templated addition of adenine by Taq DNA polymerase implications for PCR based genotyping and cloning Biotechniques 21 700 709 Mansfield E S Robertson J M Vainer M Isenberg A R Frazier R R Ferguson K Chow S Harris D W Barker D L Gill Budowle B McCord 1998 Analysis of multiplexed short tandem repeat STR systems using capillary array electrophoresis Electrophoresis 19 101 107 Mills K A Even D and Murrau J C 1992 Tetranucleotide repeat polymorphism at the human alpha fibrinogen locus FGA Hum Mol Genet 1 779 Moller A and Brinkmann B 1994 Locus ACTBP2 SE33 Sequencing data reveal considerable polymorphism Int Leg Med 106 262 267 Moller A and Brinkmann B 1995 PCR VNTRs PCR Variable Number of Tandem Repeats in forensic science Cellular amp Molec Bio 41 5 715 724 Momhinweg E Luckenbach C Fimmers R and Ritter H 1998 D3S1358 sequence analysis and gene frequency in a G
61. g GeneMapper D X Software v1 0 1 or higher to perform human identification HID analysis with AmpF STR kits be aware that HID analysis requires at least one allelic ladder sample per run folder Your laboratory can use multiple ladder samples in an analysis provided that you conduct the appropriate validation studies For multiple ladder samples the GeneMapper D X Software calculates allelic bin offsets by using an average of all ladders that use the same panel within a run folder Allelic ladder samples in an individual run folder are considered to be from a single run When the software imports multiple run folders into a project only the ladder s within their respective run folders are used for calculating allelic bin offsets and subsequent genotyping Allelic ladder samples must be labeled as Allelic Ladder in the Sample Type column in a project Failure to apply this setting for ladder samples results in failed analysis Injections containing the allelic ladder must be analyzed with the same analysis method and parameter values that are used for samples to ensure proper allele calling e Alleles that are not in the AmpF STR Allelic Ladders do exist Off ladder OL alleles may contain full and or partial repeat units An off ladder allele is an allele that occurs outside the 0 5 nt bin window of any known allelic ladder allele or virtual bin G 5 D 5 Ke e gt 120 e
62. ic Ladder Mark Sample for Deletion 70 410 190 270 310 350 230 B 1200 800 400 9170009000 Allelic_Ladder_HO2_016 fsa Mark Sample for Deletion 70 110 150 130 230 270 310 350 Figure 1 GeneMapper D X Software plot of the AmpF STR NGM Kit Allelic Ladder 12 PCR Amplification Kit User s Guide Product overview Control DNA 007 Figure 2 shows amplification of Control DNA 007 using the AmpF STR NGM profile Kit 2009 04 14_NGM_1ng_A01fsa NGM_ing Mark Sample Deletion 2009 04 14_NGM_1ng_A01fsa NGM_1ng Mark Sample Deletion 70 110 150 130 230 270 310 350 2009 04 14 NGM 1ng A01fsa NGM 1na m Mark Sample For Deletion 70 110 150 130 230 270 310 350 B B m Lis bs be 2009 04 14_NGM_1ng_A01 fsa NGM 1ng Mark Sample for Deletion 70 110 150 130 230 270 310 350 Figure 2 1 of Control DNA 007 amplified with the AmpF STR NGM Kit and analyzed on the Applied Biosystems 3130x Genetic Analyzer AmpF amp STR PCR Amplification Kit User s Guide 13 Chapter 1 Overview Workflow overview Extract DNA AutoMate Express Forensic DNA Extraction System with PrepFiler Express Forensic DNA Extra
63. ican Association of Blood Banks 58 pp Barber M D Piercy R C Andersen J F and Parkin 1995 Structural variation of novel alleles at the Hum vWA and Hum FES FPS short tandem repeat loci Int J Leg Med 108 31 35 Barber M D and Parkin B H 1996 Sequence analysis and allelic designation of the two short tandem repeat loci D18S51 and D8S1179 Intl J Legal Med 109 62 65 Barber M D McKeown B J and Parkin B H 1996 Structural variation in the alleles of a short tandem repeat system at the human alpha fibrinogen locus Int J Leg Med 108 180 185 Baron H Fung S Aydin A Bahrig S Luft F C Schuster 1996 Oligonucleotide ligation assay for the diagnosis of familial hypercholesterolemia Nat Biotechnol 14 1279 1282 Begovich A B McClure G R Suraj VC Helmuth R C Fildes N Bugawan T L Erlich H A Klitz W 1992 Polymorphism recombination and linkage disequilibrium within the HLA class II region J Immunol 148 249 58 Bender K Farfan M J Schneider 2004 Preparation of degraded human DNA under controlled conditions Forensic Sci Int 139 134 140 Bonferroni C E 1936 Teoria statistica delle classi e calcolo Belle probabilita Publicazioni del R Istituto Superiore di Scienze Economiche e Commerciali di Firenze 8 3 62 Brinkman B Klintschar M Neuhuber Huhne J and Rolf B 1998 Mutation rate in human microsatellites Influence of the stru
64. in The AmpF STR NGM Kit delivers a 16 locus multiplex with a greater power of discrimination better sensitivity and improved robustness than earlier generation kits The kit uses modified PCR cycling conditions for enhanced sensitivity a new buffer formulation to improve performance with inhibited samples more loci concentrated in the low molecular weight region of the profile to improve performance on degraded samples and an improved process for synthesis and purification of the amplification primers to deliver a much cleaner electrophoretic background AmpF STR NGM Kit contains all the necessary reagents for the amplification of human genomic DNA The reagents are designed for use with the following Applied Biosystems instruments Applied Biosystems 3500 3500xL Genetic Analyzer ABI PRISM 3100 3100 Avant Genetic Analyzer Applied Biosystems 3130 3130x Genetic Analyzer Applied Biosystems 310 Genetic Analyzer GeneAmp PCR System 9700 with the Silver 96 Well Block GeneAmp PCR System 9700 with the Gold plated Silver 96 Well Block The AmpF STR NGM Kit employs the latest improvements in primer synthesis and purification techniques to minimize the presence of dye labeled artifacts These improvements result in a much cleaner electropherogram background that enhances the assay s signal to noise ratio and simplifies the interpretation of results AmpF amp TR NGM PCR Amplification Kit User s Guide
65. ion in laboratory temperature can affect fragment migration speed and result in sizing variation Applied Biosystems recommends the following frequency of allelic ladder injections this frequency should account for normal variation in run speed However during internal validation studies verify the required allelic ladder injection frequency to ensure accurate genotyping of all samples in your laboratory environment When genotyping it is critical to use an allelic ladder run under the same conditions as the samples because Size values obtained for the same sample can differ between instrument platforms because of different polymer matrices and electrophoretic conditions Variation in laboratory temperature can affect migration speed see IMPORTANT above These variations can result in sizing variations between both single and multiple capillary runs with a greater size variation between those samples injected in multiple capillary runs than between those samples injected in a single capillary run 34 AmpF4STR9 PCR Amplification Kit User s Guide Section 3 1 Electrophoresis Section 3 1 3100 3100 Avant and 3130 3130x instruments Set up the 3100 3100 Avant or 3130 3130x instrument for electrophoresis Reagents and 3100 31 00 Avant or 3130 31 30 instrument parts Appendix B Ordering Information on page 89 lists the required materials not supplied with the AmpF STR NGM PCR Amplification Kit
66. ion of new miniSTR loci to aid analysis of degraded DNA Forensic Sci 50 43 53 DeFranchis R Cross N C P Foulkes N S and Cox T M 1988 A potent inhibitor of Taq DNA polymerase copurifies with human genomic DNA Nucleic Acids Res 16 10355 Drabek J Chung D T Butler J M McCord 2004 Concordance study between Miniplex assays and a commercial STR typing kit Forensic Sci 49 859 860 Edwards A Civitello A Hammond H and Caskey 1991 DNA typing genetic mapping with trimeric and tetrameric tandem repeats Am Hum Genet 49 746 756 Edwards A Hammond Lin J Caskey and Chakraborty 1992 Genetic variation at five trimeric and tetrameric tandem repeat loci in four human population groups Genomics 12 241 253 Farfan M J Sanz P Lareu M V and Carracedo A 9 30 1999 Population data on the D1S1656 and D12S391 STR loci in Andalusia south Spain and the maghreb north Africa Forensic Sci Int 104 1 33 36 Federal Bureau of Investigation DNA Advisory Board 1998 Quality Assurance Standards for Forensic DNA Testing Laboratories Washington DC Federal Bureau of Investigation Frank W Llewellyn B Fish P etal 2001 Validation of the AmpF STR Profiler Plus PCR Amplification Kit for use in forensic casework Forensic Sci 46 642 646 Gamero J J Romero J L Gonzalez J L Arufe M L Cuesta M I Corte Real E Carvalho
67. ired volumes of components into an appropriately sized polypropylene tube Vortex the tube then centrifuge briefly Into each well of a MicroAmp Optical 96 Well Reaction Plate add 10 uL of the formamide size standard mixture b 1 uL of PCR product or allelic ladder Note For blank wells add 11 uL of Hi Di Formamide Seal the reaction plate with appropriate septa then centrifuge the plate to ensure that the contents of each well are collected at the bottom Heat the reaction plate in a thermal cycler for 3 minutes at 95 C Immediately place the plate on 1 for 3 minutes Prepare the plate assembly then place onto the autosampler Ensure that a plate record is completed and link the plate record to the plate Start the electrophoresis run AmpF amp TR NGM PCR Amplification Kit User s Guide Chapter 3 Electrophoresis Section 3 2 3500 3500xL Series instruments Set up the 3500 3500xL instrument for electrophoresis Reagents and 3500 instrument requirements parts Appendix B Ordering Information on page 89 lists the required materials not supplied with the AmpF STR NGM PCR Amplification Kit IMPORTANT The fluorescent dyes attached to the primers are light sensitive Protect the AmpF STR NGM Primer Set from light when not in use Amplified DNA AmpF STR NGM Allelic Ladder and GeneScan 500 LIZ Size Standard v2 0 should also be protected from light Keep freeze thaw cycles to a
68. ize standard indicated in the table is a suggested amount Determine the appropriate amount of size standard based on your results and experiments Pipet the required volumes of components into an appropriately sized polypropylene tube Vortex the tube then centrifuge briefly Into each well of a MicroAmp Optical 96 Well Reaction Plate or each MicroAmp optical strip tube add a 10 uL of the formamide size standard mixture b 1 uL of PCR product or allelic ladder Note For blank wells add 11 uL of Hi Di Formamide Seal the reaction plate or strip tubes with the appropriate septa then centrifuge to ensure that the contents of each well are collected at the bottom Heat the reaction plate or strip tubes in a thermal cycler for 3 minutes at 95 C Immediately put the plate or strip tubes on ice for 3 minutes Prepare the plate assembly then put it onto the autosampler Ensure that a plate record is completed and link the plate record to the plate AmpF amp TR NGM PCR Amplification Kit User s Guide Prepare samples for electrophoresis the 3500 3500xL instrument 10 Start the electrophoresis run 2 E e 3 AmpF amp STR PCR Amplification Kit User s Guide 39 Section 3 2 Electrophoresis 40 AmpFSTR PCR Amplification Kit User s Guide Section 3 3 310 Instrument Chapter 3 Electrophoresis Set up the 310 instrument for electrophoresi
69. izing algorithm 75 100 139 150 160 200 300 350 400 and 450 58 AmpF4 TR PCR Amplification Kit User s Guide GeneMapper ID Software Use the following procedure to create the size standard for the AmpF STR NGM Kit 1 Select Tools GeneMapper Manager to open the GeneMapper Manager ICQ Manager x Projects Analysis Methods Table Settings Plot Settings Matrices Last Saved Owner Type Description 377 F HID GS500 2004 05 28 11 34 3 grid Basic Advanced Factory Provided CE_G5_HID_GS500 2004 05 28 11 34 3 gmid Basic Advanced Factory Provided CE_F_HID_GS500 2004 05 28 11 34 3 grid Basic Advanced Factory Provided Import Export C 0 5 0 lt 0 O S 2 Select the Size Standards tab then click New 3 Complete the Name field as shown below or with a name of your choosing In the Size Standard Dye field select Orange In the Size Standard Table enter the sizes specified in Create a HID size standard on page 58 2 5 Standard Editor Edit r Size Standard Description Name CE G5 NGM GS500 Description Size Standard Dye orange hal r Size Standard Table Size in Basepairs 100 0 138 0 150 0 160 0 200 0 300 0 350 0 EJ ES 4
70. l IPC assay that consists of an IPC template DNA a synthetic sequence not found in nature two primers for amplifying the IPC template DNA and one TaqMan MGB probe labeled with VIC dye for detecting the amplified IPC DNA Quantifiler Duo DNA Properties Quantifiler Duo DNA Quantification Kit Quantification Kit User s Manual Part no 4387746 Part no 4391294 The Quantifiler Duo Kit is highly specific for human DNA and combines the detection of both total human and male DNA in one PCR reaction The kit detects single stranded and degraded DNA How it works The Quantifiler Duo DNA Quantification Kit consists of target specific and internal control 5 nuclease assays The Quantifiler Duo kit combines two human specific assays in one PCR reaction for total human DNA and human male The two human DNA specific assays each consist of two PCR primers and a TaqMan probe The probes for the human DNA and human male DNA assays are labeled with VIC and FAM dyes respectively In addition the kit contains an internal PCR control IPC assay similar in principle to that used in the other Quantifiler kits but labeled with NED dye 24 AmpF4STR9 PCR Amplification Kit User s Guide Prepare the amplification kit reactions Prepare the amplification kit reactions 1 Calculate the volume of each component needed to prepare the reactions using the table below DNA sam
71. locks only party thermal cyclers 88 AmpF amp STR PCR Amplification Kit User s Guide Ordering Information Materials and equipment not included The tables below list optional equipment and materials not supplied with the AmpF STR NGM Kit Unless otherwise noted many of the items are available from major laboratory suppliers MLS Equipment Source Applied Biosystems 3500 3500xL Genetic Analyzer for Human Identification ABI PRISM 3100 3100 Avant Genetic Analyzer Applied Biosystems 3130 3130x Genetic Analyzer Applied Biosystems 310 Genetic Analyzer Contact your local Applied Biosystems sales representative GeneAmp PCR System 9700 with the Silver 96 Well Block N8050001 GeneAmp PCR System 9700 with the Gold plated Silver 96 Well Block 4314878 Silver 96 Well Sample Block N8050251 Gold plated Silver 96 Well Sample Block 4314443 Tabletop centrifuge with 96 Well Plate Adapters optional MLS Item Source 3500 3500xL Analyzer materials Anode buffer container ABC 4393927 Cathode buffer container CBC 4408256 POP 4 polymer 960 samples for 3500 3500xL Genetic Analyzers 4393710 4 polymer 384 samples for 3500 3500xL Genetic Analyzers 4393715 Conditioning reagent 4393718 8 Capillary array 36 cm for 3500 Genetic Analyzers 4404683 24 Capillary array 36 cm for 3500xL Genetic Analyzers 4404687 96 well retainer amp ba
72. lysisMethod_v1x 2011 02 25 09 43 gmidx 2 Select the Analysis Methods tab then click New to open Analysis Method Editor with the General tab selected 3 The figures below show the settings for each tab of the Analysis Method Editor Configure the Analysis Method Editor tab settings as shown in the figures below unless the instructions state otherwise Note The Analysis Method Editor closes when you save your settings see step 4 on page 74 To complete this step quickly do not save the analysis method until you finish entering settings in all of the tabs O D z D D gt 2 0 AmpF4STR9 PCR Amplification Kit User s Guide 69 Section 4 1 Data Analysis General tab settings Analysis Method Editor r Analysis Method Description Name NGM AnalysisMethod v2X Security Group GeneMapper ID X Security Group Description In the Name field either type the name as shown for consistency with files supplied with other AmpF STR kits or enter a name of your choosing In the Security Group field select the Security Group appropriate to your software configuration from the drop down list The Description and Instrument fields are optional 70 AmpF amp STR PCR Amplification Kit User s Guide Set GeneMapper ID X Software for data analysis Allele tab settings
73. ment and software overview on page 15 for a list of compatible instruments When using GeneMapper JD Software v3 2 1 to perform human identification HID analysis with AmpF STR kits be aware that HID analysis requires at least one allelic ladder sample per run folder Your laboratory can use multiple ladder samples in an analysis provided that you conduct the appropriate validation studies For multiple ladder samples the GeneMapper ID Software calculates allelic bin offsets by using an average of all ladders that use the same panel within a run folder Allelic ladder samples in an individual run folder are considered to be from a single run When the software imports multiple run folders into a project only the ladder s within their respective run folders are used for calculating allelic bin offsets and subsequent genotyping Allelic ladder samples must be labeled as Allelic Ladder in the Sample Type column in a project Failure to apply this setting for ladder samples results in failed analysis Injections containing the allelic ladder must be analyzed with the same analysis method and parameter values that are used for samples to ensure proper allele calling e Alleles that are not in the AmpF STR Allelic Ladders do exist Off ladder OL alleles may contain full and or partial repeat units An off ladder allele is an allele that occurs outside the 0 5 nt bin window of any known allelic ladder allele
74. mpFLSTR Kit reagents the use of low TE buffer reagents 10 mM Tris 0 1 mM EDTA pH 8 0 is recommended You can prepare the buffer as described in the procedure below or order it from Teknova Cat T0223 To prepare low TE buffer 1 Mix together 10mL of 1 M Tris HCl pH 8 0 0 2 mL of 0 5 EDTA pH 8 0 990 mL glass distilled or deionized water Note Adjust the volumes based on your specific needs 2 Aliquot and autoclave the solutions 3 Store at room temperature DNA quantification Importance of Quantifying the amount of DNA in a sample before amplification allows you to quantification determine whether or not sufficient DNA is present to permit amplification and to calculate the optimum amount of DNA to add to the reaction The optimum amount of DNA for the AmpFLSTR NGM Kit is 1 0 ng in a maximum input volume of 10 uL amplified for 29 cycles If too much DNA is added to the PCR reaction then the increased amount of PCR product that is generated can result in Fluorescence intensity that exceeds the linear dynamic range for detection by the instrument off scale data Off scale data are problematic because Quantification peak height and area for off scale peaks is not accurate For example an allele peak that is off scale can cause the corresponding stutter peak to appear higher in relative intensity thus increasing the calculated percent stutter Multicomponent analysis of
75. mplification Kit PCR 500Analysis gsp Products User Bulletin Part no 4332345 1 0 Windows e GeneScan36Avb_DyeSetG5Module ABI Prism 3100 3100 Avant Genetic 3100 Avant NT Injection condition 3 kV 5sec Analyzers Protocols for Processing Analyzer AmpF TR PCR Amplification Kit PCR GS500Analysis gsp Products User Bulletin Part no 4332345 t Applied Biosystems conducted validation studies for the AmpF STR NGM Kit using this configuration AmpF amp STR PCR Amplification Kit User s Guide 35 D 5 e 5 Section 3 1 Electrophoresis Prepare samples for electrophoresis on the 3100 3100 Avant or 3130 31 30x instrument Prepare the samples for electrophoresis on the 3100 3100 Avant or 3130 3130x instrument immediately before loading 1 10 36 Calculate the volume of Hi Di Formamide and GeneScan 500 LIZ Internal Size Standard needed to prepare the samples using the table below Reagent Volume per reaction pL GeneScan 500 LIZ Size Standard 0 5 uL Hi Di Formamide 9 5 uL Note Include additional samples in your calculations to provide excess volume for the loss that occurs during reagent transfers IMPORTANT The volume of size standard indicated in the table is a suggested amount Determine the appropriate amount of size standard based on your results experiments Pipet the requ
76. mplification of stutter product Mixed sample Interpret according to laboratory procedures Note Additional information will be provided on completion of validation Incomplete 3 A base addition n 1 nt position Addition of excess DNA to the reaction will contribute to the occurrence of incomplete 3 base addition Quantify DNA and add 1 0 ng of DNA to the reaction Repeat test Also be sure to include the final extension step of 60 C for 10 min in the PCR Signal exceeds dynamic range of instrument off scale data Ensure cycle number is optimized according to instructions on page 26 Repeat PCR amplification using fewer PCR cycles or use your laboratory s SOP to analyze off scale data Poor spectral separation bad matrix Follow the steps for creating a spectral file Confirm that Filter Set G5 modules are installed and used for analysis Too much DNA in reaction Use recommended amount of template DNA 1 0 ng at 29 cycles 500 pg at 30 cycles Incomplete denaturation of double stranded DNA Use the recommended amount of Hi Di Formamide and perform heat denaturation according to instructions on page 36 Poor peak height Incorrect thermal cycler parameters Check the protocol for correct thermal cycler balance parameters GeneAmp PCR System 9700 with Use Applied Biosystems GeneAmp PCR System Aluminum 96 Well block or third 9700 with silver or gold plated silver b
77. n 1 1 Mixture Analysis Quick Reference 4402094 Guide Portable document format PDF versions of this guide and the documents listed above are available at www appliedbiosystems com Note To open the user documentation available from the Applied Biosystems web site use the Adobe Acrobat Reader software available from www adobe com How to obtain support For the latest services and support information for all locations go to www appliedbiosystems com At the Applied Biosystems web site you can Access worldwide telephone and fax numbers to contact Applied Biosystems Technical Support and Sales facilities Search through frequently asked questions FAQs Submit a question directly to Technical Support Order Applied Biosystems user documents SDSs certificates of analysis and other related documents Download PDF documents Obtain information about customer training Download software updates and patches 102 PCR Amplification Kit User s Guide Bibliography Akane A Matsubara K Nakamura H Takahashi S and Kimura K 1994 Identification of the heme compound copurified with deoxyribonucleic acid DNA from bloodstains a major inhibitor of polymerase chain reaction PCR amplification J Forensic Sci 39 362 372 American Association of Blood Banks 2004 Guidance for Standards for Parentage Relationship Testing Laboratories 7th ed Bethesda Md Amer
78. ncentration to 500 pg Internal validation studies to evaluate all aspects of kit performance are required for each individual cycle number intended for operational use within the laboratory 2 Load the plate or tubes into the thermal cycler and close the heated cover IMPORTANT If using adhesive clear film instead of caps to seal the plate wells be sure to place a MicroAmp compression pad Part no 4312639 on top of the plate to prevent evaporation during thermal cycling 3 Start the run 4 On completion of the run store the amplified DNA and protect from light 26 AmpFSTR PCR Amplification Kit User s Guide Amplification using bloodstained FTA cards If you are storing the DNA Then place at lt 2 weeks 2 to 8 C gt 2 weeks 15 to 25 C IMPORTANT Store the amplified products so that they are protected from light Amplification using bloodstained FTA cards 44007 36007 32007 28007 20007 16007 12007 8007 4007 110 FTA cards can be useful for the collection storage and processing of biological samples A small punch disc of the card containing the sample can be placed directly into an amplification tube purified and amplified without transferring the disc Applied Biosystems studies indicate that a 1 2 mm bloodstained disc contains approximately 5 20 ng DNA An appropriate cycle number for this high quantity of DNA is 24 cycles determin
79. nderstand the Safety Data Sheets SDSs provided by the manufacturers of the chemicals in the waste container before you store handle or dispose of chemical waste Provide primary and secondary waste containers A primary waste container holds the immediate waste A secondary container contains spills or leaks from the primary container Both containers must be compatible with the waste material and meet federal state and local requirements for container storage Minimize contact with chemicals Wear appropriate personal protective equipment when handling chemicals for example safety glasses gloves or protective clothing For additional safety guidelines consult the SDS Minimize the inhalation of chemicals Do not leave chemical containers open Use only with adequate ventilation for example fume hood For additional safety guidelines consult the SDS Handle chemical wastes in a fume hood After emptying a waste container seal it with the cap provided Dispose of the contents of the waste tray and waste bottle in accordance with good laboratory practices and local state provincial or national environmental and health regulations Waste disposal If potentially hazardous waste is generated when you operate the instrument you must e 96 Characterize by analysis if necessary the waste generated by the particular applications reagents and substrates used in your laboratory Ensure the health and safety of all pe
80. ngth During data collection on the Applied Biosystems and ABI PRISM instruments the fluorescence signals are separated by diffraction grating according to their wavelengths and projected onto a charge coupled device CCD camera in a predictably spaced pattern The 6 FAM dye emits at the shortest wavelength and it is displayed as blue followed by the VIC dye green NED dye yellow PET dye red and LIZ dye orange Although each of these dyes emits its maximum fluorescence at a different wavelength there is some overlap in the emission spectra between the dyes Figure 3 The goal of multicomponent analysis is to correct for spectral overlap Dyes 6 FAM VIC NED PET LIZ Normalized Emission Wavelength nm Figure 3 Emission spectra of the five dyes used in the AmpF STR NGM Kit AmpF amp STR PCR Amplification Kit User s Guide Materials and equipment Kit contents and storage reaction volume Materials and equipment The AmpF STR NGM PCR Amplification Kit contains materials sufficient to perform 200 Part no 4415020 or 1000 Part 4415021 amplifications at a 25 IMPORTANT The fluorescent dyes attached to the primers are light sensitive Protect the primer set from light when not in use Amplified DNA AmpF STR NGM Allelic Ladder and GeneScan 500 LIZ Size Standard should also be protected from light Keep freeze thaw cycles to a minimum Table 3 Kit
81. ns To import the AmpF STR NGM Kit panel and bin set from the Applied Biosystems web site into the GeneMapper ID Software v3 2 1 database 1 Download and open the file containing panels and bins a From the Support menu of www appliedbiosystems com select Support gt Software Downloads Patches amp Updates GeneMapper ID Software 3 2 Updates amp Patches and download the file NGM Analysis Files GMID b Unzip the file 2 Start the GeneMapper JD Software then log in with the appropriate user name and password IMPORTANT For logon instructions refer to the GeneMapper ID Software Version 3 1 Human Identification Analysis User Guide Part no 4338775 3 Select Tools gt Panel Manager 4 Find then open the folder containing the panels bins and marker stutter a Select Panel Manager in the navigation pane Panel Manager File Edit Bins View Ci m mimi im E Panel Manager Highlight this b Select File Import Panels to open the Import Panels dialog box AmpF4STR9 PCR Amplification Kit User s Guide 49 O 5 0 EV 0 e Chapter 4 Data Analysis c Navigate to then open the NGM Analysis Files GMID folder that you unzipped in step 1 on page 49 5 Select NGM_panel_v2 then click Import Note Importing this file creates a new folder in the navigation pane of the Panel Manager Am
82. od v2 INGM_panel_y2 G5 NGM 65500 2009 01 20 C ABI3130 Cerebus_012009 IB_0006 Sample NGM_AnalysisMethod_v2 NGM panel v2 CE G5 NGM 55500 2009 01 20 C ABI3130 q 0 7 Cerebus_012009 0007 Sample NGM_AnalysisMethod_v2 NGM panel v2 G5 NGM 500 2009 01 20 C ABI3130 2 8 Cerebus 012009 0008 Sample AnalysisMethod v2 NGM panel v2 G5 NGM 65500 2009 01 20 C ABI3130 2 9 Cerebus 012009 0009 Sample AnalysisMethod v2 NGM panel v2 CE G5 NGM GS500 2009 01 20 C ABI3130 D 10 Cerebus_012009 IB_0010 Sample NGM AnalysisMethod v2 _ _ 2 G5 NGM 55500 2009 01 20 C ABI3130 E 11 Cerebus 012009 0011 Sample NGM AnalysisMethod v2 NGM panel v2 G5 NGM 65500 2009 01 20 C ABI3130 12 012009 0012 Sample NGM_AnalysisMethod_v2 INGM_panel_y2 CE_G5_NGM_GS500 2008 01 20 130 S 13 Cerebus 012009 0013 Sample NGM AnalysisMethod v2 panel v2 G5 NGM 65500 2009 01 20 C ABI3130 14 Cerebus 012009 IB_0014 Sample NGM AnalysisMethod v2 NGM panel v2 G5 NGM 55500 2009 01 20 C ABI3130 gp 15 Cerebus 012009 50061 Allelic Ladder AnalysisMethod v2 NGM panel v2 CE G5 NGM 65500 2009 01 20 C ABI3130 2 Ju 0 w Sto Examine and edit a project You can display electropherogram plots from the Samples and Genotypes ta
83. off scale data 15 not accurate and it results in poor spectral separation pull up Incomplete A nucleotide addition AmpF4STR9 PCR Amplification Kit User s Guide 23 Chapter 2 Amplification When the total number of allele copies added to the PCR is extremely low allelic dropout can occur resulting in a partial profile Methods of Applied Biosystems provides several kits for quantifying DNA in samples See the quantifying DNA references cited in the following table for details about these kits Product Description References Quantifiler Human DNA Properties Quantifiler Human DNA Quantification Kit The Quantifiler Human and Quantifiler Y Quantification Kits User s Manual Part 13438 5 Human Male Kits are highly specific for human Part no 4344790 and DNA and they detect total human or male DNA Quantifilere Y Human Male respectively The kits detect single stranded and DNA Quantification Kit degraded DNA Part no 4343906 How they work The Quantifiler DNA Quantification Kits consist of target specific and internal control 5 nuclease assays The Quantifiler Human and Quantifiler Y Human Male Kits contain different target specific assays human DNA or human male DNA respectively that each consist of two locus specific PCR primers and TaqMan MGB probe labeled with dye for detecting the amplified sequence The kits each contain a separate internal PCR contro
84. oject in the Save Project dialog box then click OK to start analysis The status bar displays the progress of analysis as both completion bar extending to the right with the percentage completed indicated With text messages on the left The table displays the row of the sample currently being analyzed in green or red if analysis failed for the sample The Genotypes tab becomes available after analysis 60 PCR Amplification Kit User s Guide For more information JGeneMapper ID v3 2 1 NGM Population Data gmid Is Logged In File Edit Analysis View Tools Help eia mm S lL o EE Project Samples Genotypes 720090 20 Status Sample File Sample Sample Type Analysis Method Panel Size Standard Run Name Instrument Type fi 1 Cerebus 012009 0001 Sample AnalysisMethod v2 NGM panel v2 CE G5 NGM 05500 2009 01 20 C ABI3130 2 Cerebus 012009 IB_0002 Sample NGM AnalysisMethod v2 panel v2 CE G5 NGM 55500 2009 01 20 C ABI3130 q 3 Cerebus 012009 IB_0003 Sample NGM AnalysisMethod v2 panel v2 CE G5 NGM 65500 2009 01 20 C ABI3130 4 Cerebus 012009 0004 Sample NGM AnalysisMethod v2 NGM panel v2 G5 NGM 65500 2009 01 20 C ABI3130 5 012009 0005 Sample NGM AnalysisMeth
85. overview 14 Instrument and software overview 15 Materials and equipment 1 17 Chapter 2 PCR Amplification 21 PGR WOrk areas s eub vui cp peel ed 22 Required user supplied materials and reagents 23 DNA quantification cc ena Rm ER ee ea 23 Prepare the amplification kit reactions 25 Perform zx uu es usd aaa NA RA 26 Amplification using bloodstained FTA cards 27 Chapter 3 Electrophoresis 33 Allelic ladder requirements 34 Section 3 1 3100 3100 Avant and 3130 3130x instruments 35 Set up the 3100 31 00 Avant or 3130 3130x instrument for electrophoresis 35 Prepare samples for electrophoresis on the 3100 3100 Avant or 3130 3130xl instrument 36 Section 3 2 3500 3500xL Series instruments 37 Set up the 3500 3500xL instrument for electrophoresis 37 Prepare samples for electrophoresis on the 3500 3500xL instrument 38 Section 310 Instrument 41 Set up the 310 instrument for electrophoresis 41 Prep
86. pFLSTR_NGM_v2 This folder contains the panel and associated markers f Import Panels Look in NGM Analysis Files GMID hd 1 NGM_bins_v2 txt File name panel v2txt Import Files of type m Files lt Cancel 6 Import NGM bins v2 a Select the AmpFLSTR v2 folder in the navigation pane File Edit Bins View 111 anel Name Comment I Bin set P 1 IGM_panel_v2 null EE Panel Manager AmpFLSTR_Panels_v1 AmpFLSTR_NGM_v2 b Select File gt Import Bin Set to open the Import Bin Set dialog box c Navigate to then open the NGM Analysis Files GMID folder d Select NGM_bins_v2 then click Import Note Importing this file associates the bin set with the panels in the NGM_panel_v2 folder 50 AmpF amp STR PCR Amplification Kit User s Guide GeneMapper ID Software C analysis Files GMD y eves NGM_bins_v2 txt NGM_panel_v2 txt Look in Highlight this File name My Documents INGM_bins_v2b Import Files of type Files 7 View the imported panels the navigation pane a Double click the AmpFLSTR v2 folder to view the NGM panel v2 folder b Double click the panel v2 folder to display the panel information in the right pane le Edit Bins View JPanel Manager x
87. ple Volume per reaction pL AmpF STR NGM Master Mix 10 0 HL AmpFLSTR NGM Primer Set 5 0 HL Note Include additional reactions in your calculations to provide excess volume for the loss that occurs during reagent transfers 2 Prepare reagents Thaw AmpFLSTR NGM Master Mix and the AmpF STR NGM Primer Set then vortex the tubes for 3 seconds and centrifuge them briefly before opening IMPORTANT Thawing is required only during first use of the kit After first use reagents are stored at 2 8 C and therefore do not require subsequent thawing Do not refreeze the reagents 3 Pipet the required volumes of components into an appropriately sized polypropylene tube 4 Vortex the reaction mix for 3 seconds then centrifuge briefly 5 Dispense 15 uL of reaction mix into each reaction well of a MicroAmp Optical 96 Well Reaction Plate or each MicroAmp tube 6 Prepare the DNA samples DNA sample To prepare Negative control Add 10 uL of low TE buffer 10mM Tris 0 1mM EDTA pH 8 0 Test sample Dilute a portion of the test DNA sample with low TE buffer so that 1 0 ng of total DNA is in a final volume of 10 uL Add 10 uL of the diluted sample to the reaction mix Positive control Add 10 uL of 007 control DNA 0 1 ng uL to provide 1 0 ng of total DNA in the positive control reaction The final reaction should be 25 uL 7 Seal the MicroAmp Optical 96 Well Reaction Plate wi
88. r based on SQ and CGQ only 110217 Mulligan Ref Plate19 3130xl Control Quality per project based on sample SOS SSPK MIX OMR SQ Control Type Total of Samples All thresholds met One or more thresholds not met Positive Control 0 0 0 Custom Control 0 0 INegative Control 0 0 Total 0 0 0 0 0 Sample Quality per project based on sample PQVs SOS SSPK MIX OMR SQ CGQ Total of Samples All thresholds met One or more thresholds not met 11 Samples 16 s Examine and edit You can display electropherogram plots from the Samples and Genotypes tabs of the a project Project window to examine the data These procedures start with the Analysis Summary tab of the Project window assuming the analysis is complete For more information For quick set up instructions refer to the GeneMapper ID X Software Version 1 0 Getting Started Guide Part no 4375574 For details about GeneMapper Software features allele filters peak detection algorithms and project editing refer to GeneMapper ID X Sofiware Version 1 0 Getting Started Guide Part no 4375574 GeneMapper ID X Sofiware Version 1 0 Quick Reference Guide Part no 4375670 GeneMapper ID X Sofiware Version 1 0 Reference Guide Part no 4375671 GeneMapper ID X Software Version 1 1
89. rder Least Squares sizing method in combination with the GeneScan 500 LIZ size standard Alternative sizing methods should be selected only after extensive evaluation as part of an internal validation study in the user s laboratory PCR Amplification Kit User s Guide Set GeneMapper ID X Software for data analysis Normalization a Normalization checkbox is available on this tab in GeneMapper 0 Software v1 2 or higher for use in conjunction with data run on the Applied Biosystems 3500 Series Genetic Analyzers Users of this version of software should perform laboratory evaluations to determine whether to use the Normalization feature for analysis of NGM kit data Peak Quality tab settings Analysis Method Editor General Allele Peak Detector Peak Quality SQ amp GQ Settings Min Max Peak Height LPH MPH Homozygous min peak height TBD Heterozygous min peak height TBD Max Peak Height MPH TBD Peak Height Ratio PHR Min peak height ratio Broad Peak BD Max peak width basepairs Allele Number AN Max expected alleles Allelic Ladder Spike Spike Detection Cut off Value IMPORTANT To be determined TBD indicates values to be determined in your laboratory Laboratories must perform the appropriate internal validation studies to determine the minimum heterozygous and homozygous minimum pe
90. rsonnel in your laboratory AmpF amp TR NGM PCR Amplification Kit User s Guide Chemical waste safety Ensure that the instrument waste 15 stored transferred transported and disposed of according to all local state provincial and or national regulations IMPORTANT Radioactive or biohazardous materials may require special handling and disposal limitations may apply AmpF amp STR PCR Amplification Kit User s Guide 97 Appendix Safety Biological hazard safety General WARNING BIOHAZARD Biological samples such as tissues body fluids biohazard infectious agents and blood of humans and other animals have the potential to transmit infectious diseases Follow all applicable local state provincial and or national regulations Wear appropriate protective equipment which includes but is not limited to protective eyewear face shield clothing lab coat and gloves All work should be conducted in properly equipped facilities using the appropriate safety equipment for example physical containment devices Individuals should be trained according to applicable regulatory and company institution requirements before working with potentially infectious materials Read and follow the applicable guidelines and or regulatory requirements in the following Department of Health and Human Services guidelines published in Biosafety in Microbiological and Biomedical Laboratories stock no 017 040 0054
91. s Reagents and Appendix Ordering Information on page 89 lists the required materials not 310 instrument requirements parts supplied with the AmpF STR NGM PCR Amplification Kit IMPORTANT The fluorescent dyes attached to the primers are light sensitive Protect the AmpF STR NGM Primer Set from light when not in use Amplified DNA AmpF STR NGM Allelic Ladder and GeneScan 500 LIZ Size Standard should also be protected from light Keep freeze thaw cycles to a minimum The following table lists Data Collection Software and the run modules that can be used to analyze AmpF STR NGM Kit PCR products For details on the procedures refer to the documents listed in the table Data Collection 5 d Run modules and conditions References Software y 3 1 Windows e GS STR POP4 1mL G5 ABI PRISM 310 Genetic Analyzer User s Manual or or v2 md5 Windows Part no 4317588 3 01 Windows Injection condition ABI PRiSM 310 Protocols for Processing NT and 15 kV 5 sec AmpF amp TR PCR Amplification Kit Products with Windows Microsoft Windows NT Operating System User 2000 Bulletin Part no 4341742 t Applied Biosystems conducted concordance studies for the AmpF STR NGM Kit using this configuration AmpF4STR9 PCR Amplification Kit User s Guide 41 wo 5 e 3 5 gt Section 3 3 Electrophoresis Prepare samples for electrophoresis on the 310 instr
92. se set Standard 3500 3500xL Genetic Analyzers 4410228 8 Tube retainer amp base set Standard for 3500 3500xL Genetic Analyzers 4410231 8 Strip Septa for 3500 3500xL Genetic Analyzers 4410701 96 Well Septa for 3500 3500xL Genetic Analyzers 4412614 Septa Cathode Buffer Container 3500 series 4410715 Note For a complete list of parts and accessories for the 3500 3500xL instrument refer to the Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Part no 4401661 AmpF STR NGM PCR Amplification Kit 200x 1000x 4415020 4415021 AmpF4STR9 PCR Amplification Kit User s Guide 89 Appendix Ordering Information Item Source 3100 3100 Avant Analyzer materials 96 Well Plate Septa 4315933 Reservoir Septa 4315932 3100 3100 Avant Genetic Analyzer Capillary Array 36 cm 4333464 POP 4 Polymer for 3100 3100 Avant Genetic Analyzers 4316355 3100 3100 Avant Genetic Analyzer Autosampler Plate Kit 96 well 4316471 GeneScan 500 LIZ Size Standard 4322682 Running Buffer 10x 402824 DS 33 Matrix Standard Kit Dye Set G5 4345833 MicroAmp Optical 96 Well Reaction Plate N8010560 250 Glass Syringe array fill syringe 4304470 5 0 mL Glass Syringe polymer reserve syringe 628 3731 Note For a complete list of parts and accessories for the 3100 instrument refer to Appendix of the ABI PRISM 3100 Genetic Analyzer and 3100 Avant Genetic Analyzer User Reference
93. tem and may affect the reproductive system WARNING CHEMICAL HAZARD POP 4 Polymer for 3100 3100 Avant Genetic Analyzers is irritating to eyes respiratory system and skin It causes adverse cardiovascular effects It contains a known or suspected reproductive toxin and a known or suspected mutagen WARNING CHEMICAL HAZARD POP 7 Polymer for the 3730 Genetic Analyzer is harmful by inhalation and if swallowed and irritating to eyes respiratory system and skin AmpF4STR9 PCR Amplification Kit User s Guide 99 Appendix Safety 100 AmpFSTR PCR Amplification Kit User s Guide Documentation Related documentation For additional documentation see How to obtain support on page 102 Document title biel ABI 5 3100 3100 Avant Data Collection v2 0 User Guide 4347102 ABI Prisu 3100 3100 Avant Genetic Analyzers Using Data Collection 4350218 Software v2 0 User Bulletin ABI PRISM 3100 Genetic Analyzer User Manual Data Collection v1 1 4315834 ABI Prism 3100 3100 Avant Genetic Analyzers Protocols for Processing 4332345 AmpF amp STR PCR Amplification Kit PCR Products User Bulletin AmpF4STR9 PCR Amplification Kit PCR Setup Quick Reference 4442401 Card AmpF amp STR PCR Amplification Kit CE Quick Reference Card 4442693 Applied Biosystems 3130 3100xl Genetic Analyzers Using Data Collection 4363787 Software v3 0 User Bulletin
94. th MicroAmp Clear Adhesive Film or MicroAmp Optical Adhesive Film or cap the tubes AmpF amp STR PCR Amplification Kit User s Guide 25 Chapter 2 Amplification 8 Centrifuge the tubes or plate at 3000 rpm for about 20 seconds in a tabletop centrifuge with plate holders if using 96 well plates to remove bubbles 9 Amplify the samples in a GeneAmp PCR System 9700 with the Silver 96 well block or a GeneAmp PCR System 9700 with the Gold plated Silver 96 well block Note The AmpFLSTR NGM Kit is not validated for use with the GeneAmp PCR System 9700 with the Aluminium 96 well block Use of this thermal cycling platform may adversely affect the performance of the AmpFSTRY NGM Kit Perform PCR WARNING PHYSICAL INJURY HAZARD Thermal cycler 1 Program the thermal cycling conditions IMPORTANT When using the GeneAmp PCR System 9700 with either 96 well silver or gold plated silver block select the 9600 Emulation Mode Initial Cycle 29 30 cycles Final Final incubation step extension hold Denature Anneal HOLD CYCLE HOLD HOLD 95 94 59 60 4 11 min 20 sec 10 IMPORTANT The NGM kit is validated for use at both 29 and 30 cycles The optimum conditions for the NGM kit are 29 cycles of amplification with a 1 ng input DNA concentration Laboratories choosing to use the NGM kit at 30 cycles should reduce the input DNA co
95. the foregoing patent claims for using only this amount of product solely in forensic and paternity testing including reporting results of purchaser s activities for a fee or other commercial consideration and also for the purchaser s own internal research No right under any other patent claim is conveyed expressly by implication or by estoppel For information on obtaining additional rights please contact outlicensing lifetech com or Out Licensing Life Technologies 5791 Van Allen Way Carlsbad California 92008 AmpF STR NGM PCR Amplification Kit is covered by U S Patent No 5 364 759 owned by Baylor College of Medicine and is sold under license from Baylor College of Medicine Not for re sale TRADEMARKS Trademarks of Life Technologies Corporation and its affiliated companies AB Design ABI PRISM AmpF STR Applied Biosystems GeneAmp GeneMapper Hi Di LIZ MicroAmp MiniFiler NED NGM PET 4 7 PrepFiler Quantifiler SGM Plus VIC TaqMan is a registered trademark of Roche Molecular Systems Inc Windows and Windows NT are registered trademarks of Microsoft Corporation Whatman and FTA are registered trademarks of GE Healthcare companies Part Number 4466844 Rev A 04 2011 Contents i o 7 Safety Information as 7 Chapter 1 el prec 9 Prod ct oVerVIeW cu ee n Ed eder Ge EE gd 10 Workflow
96. ument Prepare the samples for capillary electrophoresis on the 310 instrument immediately before loading 1 10 42 Calculate the volume of Hi Di Formamide and GeneScan 500 LIZ Internal Size Standard needed to prepare the samples using the table below Reagent Volume per reaction pL GeneScan 500 LIZ Size Standard 0 75 uL Hi Di Formamide 24 25 uL Note Include additional samples in your calculations to provide excess volume for the loss that occurs during reagent transfers IMPORTANT The volume of size standard indicated in the table is a suggested amount Determine the appropriate amount of size standard based on your results and experiments Pipette the required volumes of components into an appropriately sized polypropylene tube Vortex the tube then centrifuge briefly Into each 0 2 mL or 0 5 mL sample tube add a 25 uL of the formamide size standard mixture b 1 5 uL of PCR product or allelic ladder Note For blank wells add 25 uL of Hi Di Formamide Seal the tubes with the appropriate septa then briefly centrifuge to ensure that the contents of each tube are mixed and collected at the bottom Heat the tubes in a thermal cycler for 3 minutes at 95 C Immediately place the tubes on ice for 3 minutes Place the sample tray on the autosampler Ensure that an injection list is prepared Start the electrophoresis run AmpF amp TR NGM PCR Amplification Kit
97. y 50 amp GQ Settings Peak Detection Algorithm Advanced Ranges Peak Detection Analysis Sizing Peak Amplitude Thresholds v All Sizes v TAA B j Eo ae TBD TBD P TBD v TBD o TBD Smoothing and Baselining Aa Peak Half width 2 Light Polynomial Degree 3 O Heavy Peak Window Size 15 51 pts Slope Threshold Peak Start 0 0 Smoothing Baseline Window Size Calling Method 2nd Order Least Squares 3rd Order Least Squares Cubic Spline Interpolation Local Southern Method Global Southern Method Peak End 0 0 Normalization Use Normalization if applicable Factory Defaults IMPORTANT To be determined TBD indicates values to be determined in your laboratory Laboratories must perform the appropriate internal studies to determine the appropriate peak amplitude thresholds for interpretation of AmpFLSTR NGM Kit data Fields include Peak amplitude thresholds The software uses these parameters to specify the minimum peak height in order to limit the number of detected peaks Although GeneMapper Y Software displays peaks that fall below the specified amplitude in electropherograms the software does not label or determine the genotype of these peaks Size calling method NGM kit has been validated using the 3 O

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