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AmpFlSTR Sinofiler PCR Amplification Kit User Guide (PN 4384256C)

1. African American aeara 20 17 7 62 2 27 6 43 6 40 18 11 75 19 55 7 90 9 00 18 2 t 0 23 t 19 13 65 13 18 9 74 7 58 19 3 0 16 t t t 20 5 71 5 45 5 15 2 37 20 3 t t t 1 42 21 0 32 1 36 0 55 0 47 21 3 0 32 t 0 181 4 74 22 t t E 22 3 0 16 t t 0 71 23 0 48 t t 0 24 23 3 0 47 24 0 16 t t 24 3 t t 0 24 25 0 32 t t t p value 0 04168 0 22 0 33 0 06 D7S820 6 0 161 7 0 79 0 231 1 29 1 42 8 19 05 13 64 18 01 13 27 9 10 63 5 45 14 34 8 77 AmpF amp TR Sinofiler PCR Amplification Kit User Guide Population Data Table 5 5 AmpF STR Sinofiler kit allele frequencies continued African A American 020 10 32 38 18 41 28 12 27 96 10 2 0 23 t t 11 21 90 36 14 21 51 23 46 11 3 t t 0 24 12 12 70 22 73 13 24 19 19 13 2 06 1 82 2 76 4 74 14 0 32 0 68 0 55 0 95 15 t 0 23 0 18 t p value 0 53 0 23 0 18 0 37 D8S1179 8 0 161 1 65 0 95 9 0 48 0 23 1 65 0 47 10 1 90 11 82 8 09 8 06 11 3 81 11 14 7 72 6 64 12 11 27 12 5 15 62 7 82 13 19 52 20 68 31 99 29 62 14 34 13 17 73 19 30 28 67 15 20 79 14 55 10 66 13 74 16 6 98 8 86 3 12 3 55 17 0 95 1 82 0 18 0 47 18 f 0 23 t 19 t t t t 5 53 AmpFSTR Sinofiler PCR Amplification Kit User Guide Chapter 5 Experiments and Results
2. v1 b Select File Import Bin Set to open the Import Bin Set dialog box c Navigate to then open the GMID Sinofiler files folder d Select AmpFLSTR Sinofiler Bins vl then click Import Note Importing this file associates the bin set with the panels in the AmpFLSTR Sinofiler Panels vl folder 2 Import Bin Set Bin Se Look in D GMID Sinofiler files AmpFLSTR_Sinofiler_Bins_v1 AmpFLSTR_Sinofiler_Panels_v1 9 05 Sinofiler Gs500 Sinofiler_HID_v1 Desktop d File name AmpFLSTR_Sinofiler_Bins_v1 txt My Documents _ Files of type 00 AmpFSTR Sinofiler PCR Amplification Kit User Guide Setting Up GeneMapper ID Software for Analyzing AmpF4STR Sinofiler Kit Data To import panels and bin sets continued 7 View the imported panels in the navigation pane a Double click the AmpFLSTR_Sinofiler_Panels_v1 folder to view the Sinofiler_v1 folder b Double click the Sinofiler_v1 folder to display the panel information in the right pane and the markers below it 7 Panel Manager File Edit Bins View AmpF amp TR Sinofiler PCR Amplification Kit User Guide Ense Siofier Me 8 EQ AmpFLSTR Sinofiler Panels v1 Make Name Dye Color Mim Size Control Al
3. 14151617 18 1920 21222324252627 0125391 7 8 9 1011121314 15 16 17 18 1920 212223 24 252627 018551 Figure 5 4 Stutter percentages for 0195433 vWA 0125391 018551 loci AmpF amp TR Sinofiler PCR Amplification Kit User Guide 5 21 Chapter 5 Experiments and Results 17 0 16 0 15 0 14 0 13 0 iin 12 0 11 0 1 roar 5 10 0 i ENE E Hn 90 th 2 5 8 24 mL t pets a 79 eros 1 i 5 z 1 vs t 6 0 1 DM T E s dt 1 Y i ie i i i 50 8 m 6 40 m epee i d 3 0 Tias 20 i 1 0 0 0 21011121214 15 16 1718 19 20 21 2229 24 25 1617 18 19 20 2122 23 24 25 25 27 28 29 20 31 52 93 94 28 424344 45 46 47 48 49 50 51 52 0651043 Figure 5 5 Stutter percentages for 0651043 and FGA 5 22 AmpF amp TR Sinofiler PCR Amplification Kit User Guide Extra Peaks in the Electropherogram Table 5 2 Marker specific stutter percentages ratios used GeneMapper D AmpFLSTR_Sinofiler_panels_v1 for Sinofiler Kit loci Locus Stutter CSF1PO 8 5 012 391 14 5 013 317 8 016 539 10 018 51 16 019 433 13 5 021 11 10 02 1338 13 03 1358 11 05 818 7 5 06 1043 11 D7S820 10 08 1179 9 5 FGA 13 vWA 13 5 Addition of 3 A Nucleotide AmpliTaq Gold enzyme
4. r Pull up peak Pull up ratio Allele number Max expected alleles Factory Defaults 4 14 AmpF amp TR Sinofiler PCR Amplification Kit User Guide Setting Up GeneMapper ID Software for Analyzing AnpFSTR Sinofiler Kit Data Table 4 1 Sinofiler_HID_v1 Advanced Mode analysis method settings continued Tab Settings Analysis Method Editor HID Thresholds General Allele Peak Detector Peak Quality Quality Flags Quality weights are between 0 and 1 Quality Flag Settings Spectral Pull up 08 Control Concordance Broad Peak 08 Low Peak Height Out of Bin Allele 08 Off scale Overlap 08 Peak Height Ratio Thresholds Sizing Quality From 0 FromO 0to 025 Genotype Quality From 0 Fromo oto 025 Factory Defaults AmpF amp TR Sinofiler PCR Amplification Kit User Guide 4 15 Chapter 4 Analyzing Data Importing an HID Size Standard v3 2 1 and v3 3 4 16 The size standard for the AmpF STR Sinofiler PCR Amplification Kit uses the following GS500 peaks in its sizing algorithm 75 100 139 150 160 200 300 350 400 and 450 Use the following procedure to import the size standard for the Sinofiler kit from the folder that you downloaded from the Applied Biosystems web site into the GeneMapper ID software database Refer to step on page 4 4 for downloading instructions Note CE_G
5. 100 120 180 180 200 220 300 om 850 16007 is 20 20 121151415 efie 18 19 Jn 19 11 12 13 14 15 21131415 151617 1819 20 21 22 23 i a 100 120 150 150 220 2x 320 id Figure 1 1 GeneMapper D Software plot of the AmpF STR Sinofiler Allelic Ladder AmpF amp TR Sinofiler PCR Amplification Kit User Guide 1 5 Chapter1 Overview Workflow Overview Extract and Quantify DNA Quantifiler Total Human DNA Quantification Kit PCR Amplify DNA AmpF STR Sinofiler PCR Amplification Kit GeneAmp PCR System GeneAmp PCR System 9600 Thermal Cycler 9700 Thermal Cycler i Perform Electrophoresis ge a ABI PRISM ABI PRISM Applied Biosystems 310 Genetic Analyzer 3100 3100 Avant 3130 3130 Genetic Analyzer Genetic Analyzer E Analyze Data GeneMapper Software 1 6 Sinofiler PCR Amplification Kit User Guide Instrument and Software Overview Instrument and Software Overview Data Collection and Analysis Software Instrument and Software Compatibility About Multicomponent Analysis This section provides information about the data collection and analysis software versions required to run the AmpF STR Sinofiler PCR Amplification Kit on specific instruments The data collection software provides instructions to fi
6. AmpF amp TR Sinofiler PCR Amplification Kit User Guide 5 13 Chapter 5 Experiments and Results 5 14 Table 5 1 Precision results of five runs 16 capillaries run of the AmpF STR Sinofiler Allelic Ladder continued Applied Biosystems 3130x Genetic Analyzer Allele Mean Standard Deviation D7S820 6 255 26 255 68 0 053 0 082 7 259 27 259 71 0 066 0 084 8 263 32 263 74 0 058 0 089 9 267 34 267 79 0 055 0 085 10 271 36 271 82 0 06 0 101 11 275 4 275 86 0 066 0 098 12 279 43 279 9 0 063 0 09 13 283 48 283 95 0 063 0 092 14 287 52 288 0 065 0 098 15 291 57 292 06 0 068 0 106 08 1179 8 122 88 123 03 0 051 0 065 9 126 94 127 1 0 047 0 06 10 131 05 131 2 0 053 0 071 11 135 17 135 32 0 046 0 072 12 139 37 139 53 0 049 0 071 13 143 95 144 16 0 049 0 067 14 148 43 148 66 0 044 0 067 15 152 8 153 04 0 053 0 07 16 157 05 157 34 0 058 0 086 AmpF amp TR Sinofiler PCR Amplification Kit User Guide Accuracy Precision and Reproducibility Table 5 1 Precision results of five runs 16 capillaries run of the AmpF STR Sinofiler Allelic Ladder continued Applied Biosystems 3130x Genetic Analyzer Allele Mean Standard Deviation 17 161 23 161 53 0 058 0 112 18 165 33 165 64 0 059 0 098 19 169 38 169 71 0 054 0 112 FGA 17 214 44 214 82 0 068 0 094 18 218 47
7. Sinofiler PCR Amplification Kit User Guide Setting Up GeneMapper ID Software for Analyzing AnpFSTR Sinofiler Kit Data To import panels and bin sets continued 4 Find then open the folder containing the panels and bins a Select Panel Manager in the navigation pane 7 Panel Manager File Edit Bins View Highlight this Panel Manager b Select File Import Panels to open the Import Panels dialog box c Navigate to then open the GMID Sinofiler files folder that you unzipped in step 1 Select AmpFLSTR Sinofiler Panels v1 then click Import Note Importing this file creates a new folder in the navigation pane of the Panel Manager AmpFLSTR Sinofiler Panels v1 This folder contains the panels and associated markers Import Panels Look in B GMID_Sinofiler_files G AmpFLSTR_Sinotiler_Bins_v1 C AmpFLSTR Sinofiler Panels v1 e CE 05 Sinofiler GS500 2 Sinofiler t Desktop AmpFLSTR_Sinofiler_Panels_v1 txt My Documents Files of type aj Files J AmpF amp TR Sinofiler PCR Amplification Kit User Guide 4 5 Chapter 4 Analyzing Data 4 6 To import panels and bin sets continued 6 Import AmpFLSTR_Sinofiler_Bins_v1 a Select the AmpFLSTR_Sinofiler_Panels_v1 folder in the navigation pane 2 Panel Manager File Edit Bins View
8. 3 Click Analyze enter a name for the project in the Save Project dialog box then click OK to start analysis The status bar displays progress of analysis Asacompletion bar extending to the right with the percentage 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 7 GeneMapper ID v3 2 1 Untitled gmid Is Logged In Edt Analysis Tools Heb Bia concordance Be Sones Sample Nome Sample Type Specimen Cates Analysis Method Size Standard 550 Sample ot Hom 8 _ 5_ 555 Sample Si _ 5_5 555 Sample 2006 11 08 7 Sample Sample Sample Sample Sample Sample T HOV CE G5 5 5550 Sample HD S Sinofiler v CE_G5_Sinotiler_GSSOC Sane Fov Sret E GE Srofir 558 eis Laer no expo Seti Sio 6580 3 dedede de de de de dedede did Figure 4 1 Project Window before analysis For more information about any of these tasks refer to the GeneMapper ID Software Version 3 1 Human Identification Analysis User Guide PN 4338775 AmpF amp TR Sinofiler PCR Amplification Kit User Guide Analyzing
9. 1 Goto www appliedbiosystems com click Support then click MSDS Search 2 In the Keyword Search field enter the chemical name product name MSDS part number or other information that appears in the MSDS of interest then click Search 3 Find the MSDS of interest click the link or right click the MSDS title then select any of the following Open To view the MSDS AmpF amp TR Sinofiler PCR Amplification Kit User Guide vii Preface Chemical Waste viii Hazards Print Target To print the MSDS Save Target As To download a PDF version of the MSDS Note For the MSDSs of chemicals not distributed by Applied Biosystems contact the chemical manufacturer N HAZARDOUS WASTE Refer to Material Safety Data Sheets and local regulations for handling and disposal Norme CHEMICAL WASTE HAZARD Wastes produced by Applied Biosystems instruments are potentially hazardous and can cause injury illness or death Norme 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 amp Sinofiler PCR Amplification Kit User Guide
10. 5 54 Table 5 5 AmpF STR Sinofiler kit allele frequencies continued African American aeaa 20 p value 0 41 0 27 0 35 0 11 FGA 17 0 32 0 45 t 17 2 0 161 18 0 79 2 27 1 47 0 241 18 2 1 59 19 6 67 6 36 5 88 6 64 19 2 0 79 20 6 03 7 27 14 71 9 95 20 2 0 63 0 371 0 241 21 10 63 11 82 19 85 15 17 21 2 t 0 68 0 18 t 22 19 52 17 05 17 83 12 09 22 2 0 161 1 14 0 55 0 47 23 17 30 23 41 15 07 14 22 23 2 1 0 91 0 18 t 24 16 98 17 05 15 26 17 06 24 2 t 0 68 t t 25 9 05 7 05 6 80 14 45 25 2 t 0 23 t t 26 4 29 1 82 1 10 5 69 26 2 t 0 45 t t AmpF amp TR Sinofiler PCR Amplification Kit User Guide Population Data Table 5 5 AmpF STR Sinofiler kit allele frequencies continued AmpFSTR Sinofiler PCR Amplification Kit User Guide African American 20 27 2 86 0 68 0 55 2 84 28 0 95 0 23 0 18 0 71 29 0 32 0 24 30 0 16 t t 30 2 0 16 t 31 2 0 32 t 322 t t t t 33 2 t t t t 34 2 0 16 F t 42 2 t F t t 43 2 t 44 2 45 2 E 46 2 0 16 t t 47 2 t t t 48 2 t t t 50 2 51 2 t t t t p value 0 26 0 38 0 31 0 24 vWA 11 0 48 0 24 12 0 32 t 0 24 5 55 Chapter 5 Experiments and Results
11. Applied Biosystems provides several kits for accurately quantifying DNA in samples See the reference cited in Table 2 1 on page 2 5 for details about these kits AmpFSTR Sinofiler PCR Amplification Kit User Guide Table 2 1 Methods for quantifying DNA Quantifying DNA Quantification Kit PN 4343906 Quantifiler Human DNA Quantification Kit PN 4343895 Both Quantifiler kits have high specificity for human DNA The Quantifiler Y kit is highly specific for human male DNA The kit detects single stranded and degraded DNA How it works The DNA quantification assay combines two 5 nuclease assays e A target specific human DNA or human male DNA assay which consists of two primers for amplifying human or human male DNA and one TaqMan MGB probe labeled with FAM dye for detecting the amplified sequence Aninternal PCR control IPC assay which 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 dye for detecting the amplified IPC DNA Product Description References Quantifiler Y Properties Quantifiler Human Human Male DNA DNA Quantification Kits User s Manual PN 4344790 AmpFSTR Sinofiler PCR Amplification Kit User Guide 2 5 Chapter 2 PCR Amplification Preparing the Reactions 2 6 Master Mix Prepare the master mix by combining Amp
12. PCR Amplification Kit User Guide Accuracy Precision and Reproducibility Table 5 1 Precision results of five runs 16 capillaries run of the AmpF STR Sinofiler Allelic Ladder continued Applied Biosystems 3130x Genetic Analyzer Allele Mean Standard Deviation 20 190 56 190 84 0 064 0 075 21 194 46 194 71 0 053 0 085 22 198 35 198 59 0 053 0 068 23 202 2 202 45 0 055 0 076 24 206 51 206 79 0 052 0 069 AmpF amp TR Sinofiler PCR Amplification Kit User Guide 5 17 Chapter 5 Experiments and Results Extra Peaks in the Electropherogram Causes of Extra 5 18 Peaks Peaks other than the target alleles may be detected on the electropherogram Causes for the appearance of extra peaks include stutter products incomplete 3 A nucleotide addition at the n 1 position dye artifacts and mixed DNA samples see DAB Standard 8 1 2 2 Stutter Products A stutter is a well characterized PCR artifact that refers to the appearance of a minor peak one repeat unit smaller or less frequently one repeat larger than the major STR product Butler 2005 Mulero et al 2006 Sequence analysis of stutter products at tetranucleotide STR loci has revealed that the stutter product is missing a single tetranucleotide core repeat unit relative to the main allele Walsh et al 1996 The proportion of the stutter product relative to the main allele percent stutter is measured by dividing the
13. Source Applied Biosystems 3130 3100x Genetic Analyzer ABI PRISM 3100 3100 Avant Genetic Analyzer ABI PRISM 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 block 4314878 Silver 96 Well sample block N8050251 Gold plated Silver 96 Well sample block 4314443 Tabletop centrifuge with 96 well plate adapters optional Major Laboratory Supplier MLS 1 10 AmpF amp TR Sinofiler PCR Amplification Kit User Guide Materials and Equipment Table 1 3 User supplied materials Item Source AmpF STR Sinofiler PCR Amplification Kit 4382306 3130 3100x Analyzer materials 96 Well Plate Septa 4315933 Reservoir Septa 4315932 3130x 3100 Genetic Analyzer Capillary Array 36 cm 4315931 3130 3100 Avant Genetic Analyzer Capillary Array 36 cm 4333464 POP 4 Polymer for 3130 3130 Genetic Analyzers 4352755 3100 3100 Avant Genetic Analyzer Autosampler Plate Kit 96 well 4316471 GeneScan 500 LIZ Size Standard 4322682 Running Buffer 10 402824 DS 33 Matrix Standard Kit Dye Set G5 4345833 MicroAmp Optical 96 Well Reaction Plate N8010560 Hi Di Formamide 4311320 For a complete list of parts and accessories for the 3130 3130 instrument refer to Appendix A of the Applied Biosystems 3130 3130
14. 51 pts Slope Threshold Em Peak Start Polynomial Degree Size Calling Method 2nd Order Least Squares 3rd Order Least Squares Cubic Spline Interpolation 9 Local Southern Method Global Southern Method Peak End Factory Defaults The software uses the peak detection parameters to specify the minimum peak height to limit the number of detected peaks Although GeneMapper D software displays peaks that fall below the specified amplitude in electropherograms the software does not label or determine the genotype of these peaks Note The analysis range is set by the user based on the locations of the primer peaks and the size standard peaks Note For information on peak detection algorithms refer to the GeneMapper ID Software v3 1 Human Identification Analysis User Guide PN 4338775 Appendix A and the nstallation Procedures and New Features for GeneMapper ID Software v3 2 User Bulletin PN 4352543 AmpF amp TR Sinofiler PCR Amplification Kit User Guide 4 13 Chapter 4 Analyzing Data Table 4 1 Sinofiler HID v1 Advanced Mode analysis method settings continued Tab Settings Peak lit eak Qua y Analysis Method Editor HID General Allele Peak Detector B Quality Flags Signal level Homozygous min peak height Heterozygous min peak height rHeterozygote balance Min peak height ratio morphology peak width basepairs
15. AN li di us rur ped 7 8 9 10111213 14 15 16 17 18 19 20 D8S1179 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 567891011 12 1314 15 16 CSF1PO 5 6 7 8 9 10111213 141516 021511 075820 Figure 5 2 Stutter percentages for 0851179 021511 075820 and CSF1PO loci AmpF amp TR Sinofiler PCR Amplification Kit User Guide 5 19 Chapter 5 Experiments and Results 17 0 16 0 15 0 14 0 13 0 12 0 11 0 10 0 9 0 8 0 d te 7 0 1 6 0 5 0 HE 5 Percent Stutter 4 0 3 0 f 6 il i T3 2 0 Li 0 0 11121314151617181920 6 7 8 91011121314151617 7 8 910111213141516 5 6 7 8 910111213141516 14151617 18 1920 21222324252627 2829 0351358 055818 0135317 0165539 0251338 Figure 5 3 Stutter percentages for 0351358 055818 0135317 0165539 0251338 5 20 AmpF amp TR Sinofiler PCR Amplification Kit User Guide 17 0 Extra Peaks in the Electropherogram 16 0 15 0 14 0 13 0 12 0 11 0 Percent Stutter o o o ME oo 6 Cmca 68 6 9 nd oo o 7 0 6 0 i 5 0 1 40 it I 3 0 o HAM o 9 A 2 0 0 0 tt 9 1011121314 15161718 19 0195433 pe 40111213 141516 17 181920 2122232425
16. Data Collection System Software Run Module References Windows 3 0 3130 3130 HIDFragmentAnalysis36 4 1 Applied Biosystems XP Analyzer Dye Set G5 3130 3130xl Genetic Analyzers Using Data Collection Software v3 0 Protocols for Processing AmpF amp TR PCR Amplification Kit PCR Products User Bulletin PN 4363787 Windows 2 0 HiDFragmentAnalysis36 1 ABI 3100 3100 Avant 2000 Dye Set G5 Genetic Analyzers Using Data Collection Software v2 0 Protocols for Processing AmpF 4 TR PCR Amplification Kit PCR Products User Bulletin PN 4350218 Windows 1 1 8100 Analyzer Run Module ABI 3100 3100 Avant NT9 GeneScan36vb DyeSetG5Module Genetic Analyzers Protocols for Processing AmpF4STR PCR 2 Amplification Kit PCR Products User Bulletin PN 4332345 1 0 3100 Avant Run Module Analyzer GeneScan36Avb_DyeSetG5Module Analysis Module GS500Analysis gsp t Applied Biosystems conducted validation studies for the Sinofiler kit using this configuration AmpFSTR Sinofiler PCR Amplification Kit User Guide 3 3 Chapter 3 Performing Electrophoresis Preparing Samples for Electrophoresis on the 3100 3100 Avant or 3130 31 30x Instrument Preparing the Prepare the samples for electrophoresis the 3100 3100 Avant or Samples 3 130 3130x immediately before loading To prepare samples for electrophoresis l Calculate the volume of Hi Di
17. How to Obtain Support below How to Obtain Support For the latest services and support information for all locations go to www appliedbiosystems com then click the link for Support At the Support page 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 MSDSs certificates of analysis and other related documents Download PDF documents Obtain information about customer training Download software updates and patches Xii amp Sinofiler PCR Amplification Kit User Guide Overview This chapter covers Product Overview 1 2 Workflow Overview 1 6 Instrument and Software 1 7 Materials and Equipment 1 9 AmpF amp TR Sinofiler PCR Amplification Kit User Guide 1 1 Chapter 1 Overview Product Overview 1 2 Purpose Product Description About the Primers The AmpF STR Sinofiler PCR Amplification Kit Sinofiler kit is a short tandem repeat STR multiplex assay that amplifies 15 autosomal STR loci 0851179 21511 D7S820 CSF1PO 0351358 055818 0135317 0165539 0251338 0195433 vWA 0125391 018551 0651043 FGA and
18. 03 2012 Contents Preface Chapter 1 Chapter 2 Chapter 3 How to Use This Guide oafely ce seta SCR iib tee gua BARD How to Obtain More Information How to Obtain Support Overview Product OVetview rsss Workflow Overview Instrument and Software Overview Materials and Equipment PCR Amplification PORIANOFK Areas RENE LEUR IM d as eus Required User Supplied Materials and Reagents Quantifying DNA Preparing the Reactions Performing POR Amplification Using Bloodstained FTA Cards Performing Electrophoresis Allelic Ladder Requirements Setting Up the 3100 3100 Avant or 3130 3130xl Instrument for Electrophoresis Preparing Samples for Electrophoresis on the 3100 3100 Avant or 3130 3130x Instrument AmpFSTR Sinofiler PCR Amplification Kit User Guide lii Chapter 4 Chapter 5 Appendix A Bibliography Index Setting Up the 310 Instrument for Electrophoresis 3 6 Preparing Samples for Electropho
19. 24 8 1 15 23 12 13 8 53 25 6 19 5 91 10 66 6 64 26 2 54 0 91 1 65 1 66 27 0 48 t 0 37 0 24 28 t t t t p value 0 37 0 33 0 37 0 42 03 1358 9 0 48 t t 0 24 11 0 16 0 18 t 12 0 16 0 23 t 0 24 13 0 95 t 0 47 14 9 05 4 32 15 99 10 43 15 31 43 34 77 25 37 35 78 15 2 0 16 t 16 27 62 31 82 25 92 26 07 17 22 54 20 91 17 46 16 35 18 6 98 7 05 13 42 9 95 19 0 48 0 23 1 47 0 473 20 t 0 23 0 18 p value 0 08 0 00645 0 27 0 42 AmpF amp TR Sinofiler PCR Amplification Kit User Guide Population Data Table 5 5 AmpF STR Sinofiler kit allele frequencies continued African American aeaa 20 D5S818 7 0 32 3 41 0 18 4 98 8 4 92 t 0 74 0 47 9 1 27 7 05 4 78 3 08 10 7 46 20 23 4 96 4 27 11 25 08 30 91 36 40 38 15 12 34 76 22 95 33 82 31 75 13 23 97 14 09 17 83 16 35 14 1 43 0 68 0 92 0 711 15 0 79 0 23 0 37 0 24 16 t t t t p value 0 16 0 39 0 37 0 15 D6S1043 lt 8 t t 0 18 9 0 16 t 10 1 59 2 73 1 65 1 90 11 10 48 11 36 26 47 16 35 12 22 54 13 86 27 94 20 38 13 9 68 11 14 6 80 12 32 14 5 71 16 36 4 96 12 56 15 5 56 1 82 1 10 2 61 16 3 65 0 23 0 92 0 24 AmpF amp TR Sinofiler PCR Amplification Kit User Guide 5 51 Chapter 5 Experiments and Results 5 52 Table 5 5 AmpF STR Sinofiler kit allele frequencies continued
20. Component Description Volume AmpF STR PCR Two tubes containing MgCL 1 1 mL tube Reaction Mix deoxynucleotide triphosphates and bovine serum albumin in buffer with 0 05 sodium azide AmpF STR Two tubes containing fluorescently 0 55 Sinofiler Primer labeled primers and non labeled mL tube Set primers AmpF STR One tube containing 0 10 ng uL 0 3 mL Control DNA human female cell line DNA in 9947A 0 05 sodium azide and buffer refer to pages 1 3 and 1 4 for profile AmpF STR One tube of AmpF STR Sinofiler 50 uL Sinofiler Allelic Allelic Ladder containing amplified Ladder alleles See Table 1 1 on pages 1 3 and 1 4 for a list of alleles included in the allelic ladder Gold Two tubes of enzyme with an activity 50 uL tube DNA Polymerase of 5 U uL The table below lists the storage temperature for the kit components The fluorescent dyes attached to the primers are light sensitive Protect the AmpFI STR Sinofiler Primer Set from light when not in use Amplified DNA AmpF STR Sinofiler Allelic Ladder and 500 LIZ Size Standard should also be protected from light Component Storage Temperature AmpF STR PCR Reaction Mix AmpF STR Control DNA 9947A AmpF STR Sinofiler Allelic Ladder 20 C on receipt 2 to 8 C after initial use AmpliTaq Gold DNA Polymerase AmpF STR Sinofiler Primer Set 15 to
21. Formamide and GeneScan 500 LIZ Internal Size Standard needed to prepare the samples using the table below Reagent Volume 9 Per Reaction uL GeneScan 500 LIZ Size Standard 0 3 Hi Di Formamide 8 7 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 CHEMICAL HAZARD Hi Di Formamide Exposure causes eye skin and respiratory tract irritation It is a possible developmental and birth defect hazard Read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves Pipette the required volumes of components into an appropriately sized polypropylene tube Vortex the tube then centrifuge briefly 3 4 AmpFSTR Sinofiler PCR Amplification Kit User Guide Preparing Samples for Electrophoresis on the 3100 3100 Avant 3130 3130xl Instrument To prepare samples for electrophoresis continued 4 Into each well of a MicroAmp Optical 96 Well Reaction Plate add 9 uL of the formamide size standard mixture 1 uL of PCR product or Allelic Ladder Note For blank wells add 10 uL of Hi Di formamide Seal the reaction plate with appropriate septa then briefly ce
22. ID Software Version 3 1 Human Identification Analysis User Guide PN 4338775 AmpF amp TR Sinofiler PCR Amplification Kit User Guide 4 3 Chapter 4 Analyzing Data For More Info Importing Panels and Bins 8 2 1 only For quick set up instructions refer to the GeneMapper ID Software Version 3 3 Getting Started Guide PN 4385329 For details about GeneMapper ID features refer to the GeneMapper ID Sofiware Version 3 1 Human Identification Analysis User Guide PN 4338775 and the GeneMapper ID Software Versions 3 1 and 3 2 Human Identification Analysis Tutorial PN 4335523 Also refer to the Installation Procedures and New Features for GeneMapper ID Software v3 2 User Bulletin PN 4352543 To import the Sinofiler kit panels and bin sets from the Applied Biosystems web site into the GeneMapper D Software v3 2 1 database To import panels and bin sets 1 Download and open the file containing panels and bins a Openan internet browser then download the file GMID Sinofiler files zip from www appliedbiosystems com support download GeneMapper GMID_Sinofiler_files zip b Unzip the file 2 Start the GeneMapper software then log in with the appropriate user name and password IMPORTANT If you need logon instructions refer to page 2 7 of the GeneMapper ID Software Version 3 1 Human Identification Analysis User Guide PN 4338775 3 Select Tools gt Panel Manager
23. Klitz W 1992 Polymorphism recombination and linkage disequilibrium within the HLA class II region 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 Brinkman B Klintschar M Neuhuber Huhne J and Rolf 1998 Mutation rate in human microsatellites Influence of the structure and length of the tandem repeat Am Hum Genet 62 1408 1415 Brinkman Moller A and Wiegand 1995 Structure of new mutations in 2 STR systems Intl 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 AmpF amp TR Sinofiler PCR Amplification Kit User Guide Bibliography 1 Bibliography 2 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 Chen G Xin J Li Y Wu J Hou Y Li J Deng S 1999 Polymorphisms of five short tandem repeat systems in Chinese Han population in Che
24. Safety Chemical Waste minimize the hazards of chemical waste Safety Guidelines 4 Read and understand the Material Safety Data Sheets 5 55 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 MSDS 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 MSDS Handle chemical wastes in a fume hood After emptying the 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 Characterize by analysis if necessary the waste generated by the
25. When appropriate the range of DNA quantities able to produce Guideline 2 3 reliable typing results should be determined SWGDAM July 2003 Importance of optimal amount of input DNA added to the AmpF STR Quantitation Sinofiler PCR Amplification Kit should be between 0 50 and 1 25 ng The DNA sample should be quantitated prior to amplification using a system such as the Quantifiler Human DNA Quantification Kit PN 4343895 The final DNA concentration should be 0 05 to 0 125 ng uL so that 0 50 to 1 25 ng of DNA is added to the PCR reaction in a volume of 10 uL If the sample contains degraded DNA amplification of additional DNA may be beneficial In Figure 5 9 on page 5 33 the control DNA 9947A was serially diluted from 1 ng to 0 062 ng Full profiles 27 PCR products were consistently obtained at 0 125 ng but occasional partial profiles missing from 1 to 3 alleles were observed at 0 062 ng Effect of DNA If too much DNA is added to the PCR reaction the increased amount Quantity on of PCR product that is generated can result in Results Fluorescence intensity that exceeds the linear dynamic range for detection by the instrument off scale data Off scale data is a problem because Quantitation 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 calculate
26. related 1 E electropherogram causes of extra peaks 5 18 extra peaks 5 18 species specificity 5 30 electrophoresis data collection software 3 3 3 6 preparing samples 3 4 3 7 references 3 3 3 6 runmodule 3 3 3 6 setup 3 3 3 6 emission spectra 1 8 equipment not included with kit 1 10 experiments and results 5 1 extra peaks causes 5 18 Index 2 F Five Dye Analysis 1 3 to 1 4 fluorescent dyes 1 8 FTA card DNA amplification using 2 9 figure showing results 2 9 G GeneMapper ID software allele tab 4 12 analysis settings 4 17 analyzing and editing sample files 4 17 considerations 4 2 generaltab 4 11 peak detector tab 4 13 peak quality tab 4 14 quality flags tab 4 15 size standard 4 17 viewing imported panels 4 7 GeneMapper Manager 4 9 4 16 GeneScan size standard about 1 10 dyelabel 1 8 fragmentsizes 4 17 volume per reaction 3 4 3 7 guidelines chemicalsafety chemical waste disposal viii chemical waste safety 1 H hazards chemical waste viii hematin effects of 5 35 HID analysis method importing 4 9 Hi Di formamide volume per reaction 3 4 3 7 Import Panels dialog box 4 5 IMPORTANT description vi Information Development department contacting xii inheritance 5 28 Sinofiler PCR Amplification Kit User Guide italic text when to use v K kit contents 1 9 description 1 2 fluorescent dyes 1 8 instruments for use with 1 2 loci amplifie
27. 218 17 218 38 0 035 0 055 AmpF amp TR Sinofiler PCR Amplification Kit User Guide Accuracy Precision and Reproducibility Table 5 1 Precision results of five runs 16 capillaries run of the AmpF STR Sinofiler Allelic Ladder continued Applied Biosystems 3130x Genetic Analyzer Allele Mean Standard Deviation 33 220 18 220 4 0 04 0 057 33 2 222 12 222 34 0 038 0 048 34 224 25 224 47 0 048 0 058 34 2 226 16 226 39 0 042 0 056 35 228 24 228 47 0 046 0 053 35 2 230 15 230 39 0 04 0 064 36 232 14 232 39 0 044 0 053 37 236 21 236 46 0 043 0 054 38 240 15 240 38 0 049 0 059 02 1338 15 306 48 306 82 0 042 0 082 16 310 54 310 89 0 05 0 093 17 314 61 314 95 0 049 0 087 18 318 67 318 99 0 052 0 082 19 322 73 323 02 0 049 0 081 20 326 76 327 07 0 047 0 07 21 330 83 331 11 0 038 0 083 22 334 87 335 15 0 05 0 078 23 338 93 339 17 0 051 0 082 24 342 97 343 19 0 049 0 08 25 346 99 347 2 0 047 0 088 AmpFSTR Sinofiler PCR Amplification Kit User Guide Chapter 5 Experiments and Results 5 12 Table 5 1 Precision results of five runs 16 capillaries run of the AmpF STR Sinofiler Allelic Ladder continued Applied Biosystems 3130x Genetic Analyzer Allele Mean Standard Deviation 26 351 351 19 0 044 0 069 27 354 92 355 08 0 044 0 065 28 359 12 359 29 0 0
28. 24 24 2 t t t 0 24 25 t t t t 26 0 32 t 0 55 0 47 27 3 81 0 451 2 94 2 61 28 24 29 4 55 15 81 11 14 28 2 t 0 68 0 241 29 16 19 26 59 24 26 17 77 29 2 t 0 23 t 0 24 29 3 0 16 t 0 18 f 30 19 37 29 32 23 53 30 33 30 2 2 06 1 14 3 68 1 42 30 3 t 0 681 t 31 9 84 9 77 5 51 5 92 31 2 6 19 7 73 9 19 10 43 32 2 06 3 41 1 65 0 717 AmpF amp TR Sinofiler PCR Amplification Kit User Guide Population Data Table 5 5 AmpF STR Sinofiler kit allele frequencies continued African A American 20 32 2 6 98 9 77 9 56 13 51 33 0 79 1 36 0 18 0 24 33 1 0 16 t t 33 2 3 81 2 73 2 39 3 55 34 0 321 t 34 1 0 23 34 2 0 16 0 91 0 37 0 71 35 2 54 t t 0 47 35 2 t t t 36 0 79 t 0 18 t 37 38 0 16 t t p value 0 20 0 16 0 13 0 41 02 1338 lt 14 0 16 0 181 t 15 0 63 t 0 18 t 16 4 6 1 82 4 60 3 08 17 10 16 8 86 18 75 19 43 18 4 76 10 68 8 27 6 87 19 16 98 19 09 13 97 16 35 20 11 9 12 05 15 26 13 74 21 14 29 3 18 2 39 1 90 5 49 AmpFSTR Sinofiler PCR Amplification Kit User Guide Chapter 5 Experiments and Results 5 50 Table 5 5 AmpF STR Sinofiler kit allele frequencies continued African American aeara 1211 22 11 75 5 68 2 76 6 64 23 7 46 16 14 9 01 14 93
29. 25 C AmpFSTR Sinofiler PCR Amplification Kit User Guide 1 9 Chapter 1 Overview Standards for For the Sinofiler kit the panel of standards needed for PCR Samples amplification PCR product sizing and genotyping are Control DNA 9947 A positive control for evaluating the efficiency of the amplification step and STR genotyping using the AmpF STR Sinofiler Allelic Ladder GeneScan 500 LIZ Size Standard Used for obtaining sizing results It contains 16 single stranded 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 lane size standard yields precise sizing results for AmpF STR Sinofiler PCR products Order the GeneScan 500 LIZ Size Standard PN 4322682 separately 5 Sinofiler Allelic Ladder Developed by Applied Biosystems for accurate characterization of the alleles amplified by the Sinofiler kit The AmpF STR Sinofiler Allelic Ladder contains most alleles reported for the 15 autosomal loci Refer to Loci Amplified by the Kit on page 1 3 for a list of the alleles included in the Sinofiler kit Equipment and Tables 1 2 and 1 3 list required and optional equipment and materials Materials Not not supplied with the Sinofiler kit Unless otherwise noted many of Included the items are available from major laboratory suppliers MLS Table 1 2 Equipment Equipment
30. 5 61 9 100 D7S820 646 88 6 89 9 50 6 100 D8S1179 643 89 8 91 0 58 9 100 FGA 683 88 4 89 7 54 2 100 vWA 664 89 5 91 0 63 9 100 5 38 AmpF amp TR Sinofiler PCR Amplification Kit User Guide Mixture Studies If an unusually low peak height ratio is observed for one locus but there are no other indications that the sample is a mixture you can reamplify and reanalyze the sample to determine if the imbalance is reproducible Possible causes of imbalance at a locus are Degraded DNA Presence of inhibitors Extremely low amounts of input DNA A SNP in one of the primer binding sites Presence of an allele containing a rare sequence that does not amplify as efficiently as the other allele Resolution of Genotypes in Mixed Samples A sample containing DNA from two sources can be comprised at a single locus of any of the seven genotype combinations see below Heterozygote heterozygote no overlapping alleles four peaks Heterozygote heterozygote one overlapping allele three peaks Heterozygote heterozygote two overlapping alleles two peaks Heterozygote homozygote no overlapping alleles three peaks Heterozygote homozygote overlapping allele two peaks Homozygote homozygote no overlapping alleles two peaks Homozygote homozygote overlapping allele one peak Specific genotype combinations and input DNA ratios of the samples contained in a mixture deter
31. 74 0 059 0 11 21 337 49 337 81 0 067 0 107 22 341 65 341 96 0 05 0 09 23 345 63 345 92 0 059 0 091 24 349 7 349 98 0 054 0 086 25 353 65 353 9 0 063 0 086 26 357 6 357 82 0 048 0 082 27 361 54 361 76 0 042 0 073 019 433 15 101 68 101 76 0 042 0 055 16 105 56 105 64 0 039 0 054 17 109 48 109 54 0 039 0 05 18 113 4 113 47 0 034 0 052 19 115 39 115 44 0 043 0 051 20 117 35 117 4 0 037 0 044 21 119 35 119 41 0 031 0 047 22 121 32 121 36 0 035 0 048 23 123 32 123 37 0 035 0 051 amp Sinofiler PCR Amplification Kit User Guide 5 9 Chapter 5 Experiments and Results 5 10 Table 5 1 Precision results of five runs 16 capillaries run of the AmpF STR Sinofiler Allelic Ladder continued Applied Biosystems 3130x Genetic Analyzer Allele Mean Standard Deviation 24 125 3 125 35 0 027 0 048 25 127 33 127 38 0 031 0 044 26 129 33 129 36 0 035 0 042 27 131 36 131 39 0 033 0 044 28 133 36 133 41 0 04 0 055 021 11 24 184 54 184 72 0 049 0 069 24 2 186 53 186 71 0 039 0 061 25 188 49 188 67 0 039 0 067 26 192 42 192 59 0 041 0 066 27 196 38 196 54 0 036 0 047 28 200 23 200 39 0 042 0 051 28 2 202 21 202 38 0 036 0 049 29 204 2 204 36 0 042 0 046 29 2 206 24 206 42 0 041 0 049 30 208 21 208 38 0 034 0 053 30 2 210 18 210 37 0 028 0 049 31 212 22 212 38 0 036 0 052 31 2 214 18 214 37 0 042 0 061 32 216 19 216 38 0 037 0 052 32 2
32. 766 0 767 0 737 0 758 D3S1358 0 471 0 597 0 561 0 381 D5S818 0 471 0 589 0 473 0 416 D6S1043 0 690 0 694 0 665 0 682 D7S820 0 570 0 492 0 650 0 627 D8S1179 0 581 0 739 0 602 0 655 FGA 0 715 0 694 0 679 0 710 0 672 0 597 0 636 0 655 Combined 0 99999973 0 999999774 0 99999968 0 99999964 AmpF amp TR Sinofiler PCR Amplification Kit User Guide 5 61 Chapter 5 Experiments and Results The P value is the probability averaged over all possible mother child pairs that a random alleged father will be excluded from paternity after DNA typing of the Sinofiler kit STR loci Chakraborty et al 1996 5 62 AmpF amp TR Sinofiler PCR Amplification Kit User Guide Troubleshooting In This Appendix Follow the actions recommended in this appendix to troubleshoot problems that occur during analysis 2 AmpF amp TR Sinofiler PCR Amplification Kit User Guide A 1 Appendix A Troubleshooting Troubleshooting Table A 1 Troubleshooting Observation Possible Causes Recommended Actions Faint or no signal from both the AmpF STR9 Control DNA 9947A and the DNA test samples at all loci Incorrect volume or absence of AmpF STR PCR Reaction Mix AmpF STF Sinofiler Primer Set or AmpliTaq Gold DNA Polymerase Repeat amplification No activation of AmpliTaq Gold DNA Polymerase Repeat amplification
33. The other parent offspring allele transfers were in accordance with Mendelian rules as expected Mapping The Sinofiler kit loci have been mapped and the chromosomal locations have been published Nakahori et al 1991 Edwards et al 1992 Kimpton et al 1992 Kong et al 2004 Lareu et al 1996 Mills et al 1992 Sharma and Litt 1992 Li et al 1993 Straub et al 1993 Barber and Parkin 1996 AmpF amp TR Sinofiler PCR Amplification Kit User Guide 5 29 Chapter 5 Experiments and Results Species Specificity SWGDAM For techniques designed to type human DNA the potential to detect Guideline 2 2 DNA from forensically relevant nonhuman species should be evaluated SWGDAM July 2003 The AmpF STR Sinofiler PCR Amplification Kit provides the required specificity for detecting primate alleles Other species do not amplify for the loci that are tested Nonhuman Studies Nonhuman DNA may be present in forensic casework samples The data from Sinofiler kit experiments on nonhuman DNA sources are shown in Figure 5 8 30 110 130 150 170 190 210 230 250 270 290 310 330 350 Control DNA 9947A 30 110 130 150 170 130 210 230 250 270 290 310 330 350 Chimpanzee 30 110 130 150 170 190 230 310 330 350 2000 Microbial 1000 210 250 270 290 Figure 5 8 Representative electropher
34. The Calibrate function is also available in the Control Console IMPORTANT To verify your client connection to the database you need a valid user ID and password Four safety alert words appear in Applied Biosystems user documentation at points in 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 Definitions IMPORTANT Indicates information that is necessary for proper instrument operation accurate chemistry kit use or safe use ofa chemical N ize Indicates a potentially hazardous situation that 1f not avoided may result in minor or moderate injury It may also be used to alert against unsafe practices Indicates a potentially hazardous situation that if not avoided could result in death or serious injury T m T Indicates an imminently hazardous situation that if not avoided will result in death or serious injury This signal word 15 to be limited to the most extreme situations rom CHEMICAL HAZARD Some of the chemicals used with Applied Biosystems instruments and protocols are potentially hazardous and can cause injury ilIness or death AmpF STR Sinofiler PCR Amplification Kit User Guide Safety Chemical Safety minimize the hazards of chemicals Guidelines Read and understand the Material Safety Data Sheets MSDSs pro
35. and Editing Sample Files with GeneMapper ID Software Examining and You can display electropherogram plots from the Samples and Editing a Project Genotypes tabs of the Project window to examine the data These v3 2 1 and v3 3 procedures start with the Samples tab of the Project window assuming the analysis is complete For more information about any of these tasks refer to GeneMapper ID Software Version 3 3 Getting Started Guide PN 4385329 Installation Procedures and New Features for GeneMapper ID Software Version v3 2 User Bulletin PN 4352543 ID Software Version 3 1 Human Identification Analysis User Guide PN 4338775 GeneMapper ID Software Versions 3 1 and 3 2 Human Identification Analysis Tutorial PN 4335523 AmpF amp TR Sinofiler PCR Amplification Kit User Guide 4 19 Chapter 4 Analyzing Data 4 20 AmpF amp TR Sinofiler PCR Amplification Kit User Guide Part Number 4384256 Rev 03 2012 Experiments and Results This chapter covers OVERVIEW 5 2 Accuracy Precision and 5 3 Extra Peaks in the Electropherogram 5 18 Characterization 1 5 28 Species Specificity 0 0 cee cee enn 5 30 SenSILVILY POE 5 32 XR Se d 5 34 M ixt re St dies Vt sada Ce ede ve dc ede d
36. at the D128391 locus Int Legal Med 109 134 138 Lazaruk K Walsh P S Oaks F 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 electrophoresis instrument Electrophoresis 19 86 93 Li D X Bi L F Su X L 2004 Genetic polymorphism of 6 short tandem repeat loci in Mongolian population of China Zhonghua Yi Xue Yi Chuan Xue Za Zhi 21 407 409 Chinese Li H Schmidt L Wei M H Hustad T Leman M I Zbar B and Tory 1993 Three tetranucleotide polymorphisms for loci D3S1352 0351358 0351359 Hum Mol Genet 2 1327 Liu S Bi L Su X 2005 Study of genetic polymorphism of 6 short tandem repeat loci in Nongqu Mongolian of inner Mongolia Autonomous Region in China Zhonghua Yi Xue Yi Chuan Xue Za Zhi 22 222 223 Chinese Lu Zhao Q G Liu Y L Yu Y L Zhu A P Li Q Di S L Feng J Z Zhang J G Li C J 2003 Genetic polymorphism of six short tandem repeat loci in the Han population in Hebei province of China Zhonghua Yi Xue Yi Chuan Xue Za Zhi 20 259 261 Chinese Magnuson V L Ally D S Nylund S J Karanjawala Z E Rayman J B Knapp J I Lowe A L Ghosh S Collins FS 1996 Substrate nucleotide determined non templated addition of adenine by Taq DNA polymerase implications for PCR based genotyping and cloning Bi
37. database 1 76 X 104 for the U S Caucasian database and 2 57 X 10 4 for the U S Hispanic database p 1 0 where 6 0 01 Hence the minimum combined multilocus genotype frequency at 15 loci is 1 87 X 10758 for the African American database 6 15 X 10 5 for the Asian database 4 63 X 10 57 for the U S Caucasian database and 1 45 X 10 54 for the U S Hispanic database Concordance Applied Biosystems analyzed 300 samples by comparing allele calls Studies between the AmpF STR Sinofiler and AmpF STR Identifiler kits The genotype data from all the analyzed samples were concordant between the Identifiler and Sinofiler kits AmpF amp TR Sinofiler PCR Amplification Kit User Guide 5 57 Chapter 5 Experiments and Results Mutation Rate Estimation of spontaneous or induced germline mutation at genetic loci can be achieved by comparing the genotypes of offspring to those of their parents From such comparisons the number of observed mutations are counted directly In previous studies genotypes often STR loci that were amplified by the AmpF STR SGM Plus PCR Amplification Kit were determined for a total of 146 parent offspring allelic transfers meioses at the Forensic Science Service Birmingham England One length based STR mutation was observed at the D18S11 locus mutations were not detected at any of the other nine STR loci The D18S11 mutation was represented by an increase of one 4 nt repeat unit allele 17 was i
38. injected For ABI PRISM 3100 3100 Avant or 3130 3130xl instrument runs Mix 1 0 uL of PCR product and 9 uL of Hi Di Formamide GeneScan 500 LIZ9 solution N Mc CHEMICAL HAZARD Formamide causes eye skin and respiratory tract irritation It is a possible reproductive and birth defect hazard Read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves Degraded formamide Check the storage of formamide do not thaw and refreeze multiple times Try Hi Di Formamide N CHEMICAL HAZARD Formamide causes eye skin and respiratory tract irritation It is a possible reproductive and birth defect hazard Read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves AmpFSTR Sinofiler PCR Amplification Kit User Guide A 3 Appendix A Troubleshooting Table A 1 Troubleshooting continued Observation Possible Causes Recommended Actions Positive signal from AmpF STR Control DNA 99474 but partial or no signal from DNA test samples Quantity of test DNA sample is below assay sensitivity Quantitate DNA and add 0 5 to 1 25 ng of DNA Repeat test Test sample contains PCR inhibitor for example heme compounds certain dyes Quantitate DNA and add minimum necessary volume Repeat test Wash the sample in a Centricon 100 Repeat test Test sample DN
39. making sure to hold reactions initially at 95 for 11 min Master Mix not vortexed thoroughly before aliquoting Vortex the Master Mix thoroughly AmpF STR Sinofiler Primer Set exposed to too much light Store the 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 not seated tightly in the thermal cycler during amplification Push reaction tubes firmly into contact with block after first cycle Repeat test GeneAmp PCR System 9600 heated cover misaligned Align the GeneAmp 9600 heated cover properly so that white stripes align after twisting the top portion clockwise Wrong PCR reaction tube Use Applied Biosystems MicroAmp Reaction Tubes with Caps for the GeneAmp 9600 and 9700 MicroAmp Base used with tray retainer set and tubes in GeneAmp 9600 and 9700 Remove MicroAmp Base from tray retainer set and repeat test A 2 amp Sinofiler PCR Amplification Kit User Guide Table A 1 Troubleshooting continued Troubleshooting Observation Possible Causes Recommended Actions Faint or no signal from both the AmpF STR Control DNA 9947A and the DNA test samples at all loci continued Insufficient PCR product electrokinetically
40. the lower amounts of DNA analyzed using the Applied Biosystems 3130x Genetic Analyzer AmpF amp TR Sinofiler PCR Amplification Kit User Guide 5 33 Chapter 5 Experiments and Results Stability SWGDAM Guideline 2 4 5 34 The ability to obtain results from DNA recovered from biological samples deposited on various substrates and subjected to various environmental and chemical insults has been extensively documented In most instances assessment of the effects of these factors on new forensic DNA procedures is not required However if substrates and or environmental and or chemical insults could potentially affect the analytical process then the process should be evaluated using known samples to determine the effects of such factors SWGDAM July 2003 Degraded DNA As the average size of degraded DNA approaches the size of the target sequence the amount of PCR product generated is reduced due to the reduced number of intact templates in the size range necessary for amplification Degraded DNA was prepared to examine the potential for preferential amplification of loci High molecular weight Raji DNA was sonicated and incubated with increasing doses of DNase I 0 to 6 Units for 20 minutes Bender et al 2004 The DNA was examined by capillary electrophoresis analysis to determine the average size of the DNA fragments at each time point One ng of degraded DNA was amplified using the AmpFI STR Sinofiler PCR Amplif
41. the sex determining marker amelogenin in a single PCR reaction The Sinofiler 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 3130 3130x Genetic Analyzer ABI PRISM 3100 3100 Avant Genetic Analyzer ABI PRISM 310 Genetic Analyzer GeneAmp PCR System 9600 Silver 96 Well GeneAmp PCR System 9700 Gold plated silver block GeneAmp PCR System 9700 The AmpF STR Sinofiler kit employs the same primer sequences as used in the previous AmpF STR kits with the exception of 0651043 and D128391 Degenerate primers for the loci 0851179 vWA and D16S539 were added to the AmpF STR Sinofiler Primer Set to address mutations in the primer binding sites The addition of the degenerate primers allows for the amplification of those alleles in samples containing the mutations without altering the overall performance of the AmpF STR Sinofiler PCR Amplification Kit Non nucleotide linkers are used in primer synthesis for the following loci CSFIPO 055818 0135317 0165539 0251338 0125391 0185851 amelogenin and 0651043 For these primers non nucleotide linkers are placed between the primers and the fluorescent dye during oligonucleotide synthesis Butler 2005 Grossman et al 1994 and Baron et al 1996 Non nucleotide linkers enable reproducible positioning of the alleles to facili
42. to ensure accurate genotyping of all samples in your laboratory environment It is critical to genotype using 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 Slight procedural and reagent variations between single and multiple capillaries result in greater size variation than that found between samples injected in the same capillary in a single run 3 2 AmpFSTR Sinofiler PCR Amplification Kit User Guide Setting Up the 3100 3100 Avant or 3130 3130 Instrument for Electrophoresis Setting Up the 3100 3100 Avant or 3130 3130x Instrument for Electrophoresis Reagents and Electrophoresis Setup Software and Reference Parts Table 1 3 on page 1 11 lists the required materials not supplied with the AmpF STR Sinofiler PCR Amplification Kit 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 Sinofiler Allelic Ladder and GeneScan 500 LIZ Size Standard should also be protected from light Minimize freeze thaw cycles This table lists data collection software and the run modules that you can use to analyze Sinofiler kit products For details on the procedures refer to the documents listed in the table Documents Operating
43. 0 034 0 030 0 038 03 1358 0 099 0 145 0 073 0 093 05 818 0 101 0 083 0 140 0 122 D6S1043 0 028 0 034 0 057 0 029 D7S820 0 084 0 092 0 071 0 075 D8S1179 0 081 0 043 0 063 0 071 FGA 0 031 0 038 0 042 0 033 0 057 0 071 0 059 0 081 Combined 1 12 1079 8 37 x 1079 6 28 x 1079 2 31 x 1077 AmpF amp TR Sinofiler PCR Amplification Kit User Guide 5 59 Chapter 5 Experiments and Results The P value is the probability that two individuals selected at random will have an identical Sinofiler kit genotype Sensabaugh 1982 The P values for the populations described in this section are then approximately 1 8 93 X 10 5 African American 1 1 19 X 1018 Asian 1 1 59 X 1018 U S Caucasian and 1 4 33 X 1018 U S Hispanic 5 60 AmpF amp TR Sinofiler PCR Amplification Kit User Guide Probability of Paternity Exclusion Probability of Paternity Exclusion Table 5 7 shows the Probability of Paternity Exclusion values of the AmpF STR Sinofiler PCR Amplification Kit STR loci individually and combined Table 5 7 Probability of Paternity Exclusion values for the AmpF STR Sinofiler kit Locus uae Asian Caucasian Hispanic CSF1PO 0 530 0 433 0 461 0 402 D12S391 0 696 0 649 0 767 0 682 D13S317 0 397 0 556 0 535 0 583 D16S539 0 530 0 564 0 581 0 558 D18S51 0 766 0 712 0 737 0 748 D19S433 0 678 0 676 0 461 0 655 D21S11 0 741 0 676 0 723 0 673 D2S1338 0
44. 1 0138317 11 12 14 0165539 9 10 12 13 0251338 20 23 20 21 0195433 14 15 12 2 14 2 14 16 14 0125391 18 19 18 24 018551 12 15 17 19 Amelogenin X Y X 0651043 12 14 11 12 FGA 24 26 21 22 The Sinofiler kit has been optimized to reliably amplify and type approximately 0 50 to 1 25 ng of single source DNA AmpF amp TR Sinofiler PCR Amplification Kit User Guide 5 41 Chapter 5 Experiments and Results Population Data 5 42 SWGDAM Guideline 2 7 Overview The distribution of genetic markers in populations should be determined in relevant population groups SWGDAM July 2003 To interpret the significance of a match between genetically typed samples you must know the population distribution of alleles at each locus in question If the genotype of the relevant evidence sample is different from the genotype of a suspects reference sample then the suspect is excluded as the donor of the biological evidence that was tested An exclusion is independent of the frequency of the two genotypes in the population If the suspect and evidence samples have the same genotype then the suspect is included as a possible source of the evidence sample The probability that another unrelated individual would also match the evidence sample is estimated by the frequency of that genotype in the relevant population s Population Samples Used in These Studies AmpF STR Sinofiler PCR Amplification Kit was u
45. 1 8 46 3 55 24 1 90 3 18 2 76 1 18 25 1 11 1 59 0 92 1 90 26 0 16 0 23 0 55 0 71 27 0 45 0 24 28 t 0 23 t p value 0 45 0 20 0 37 0 02878 AmpF amp TR Sinofiler PCR Amplification Kit User Guide Population Data Table 5 5 AmpF STR Sinofiler kit allele frequencies continued AmpFSTR Sinofiler PCR Amplification Kit User Guide African American aeaa Meen 0138317 6 0 23 t t 7 t 0 23 t 8 3 81 29 32 9 74 8 77 9 1 59 11 82 5 70 14 93 10 3 02 15 91 7 35 9 24 11 30 48 22 73 29 78 24 17 12 44 92 14 55 30 70 27 49 13 11 90 4 09 11 76 10 43 14 3 97 0 45 4 96 4 98 15 0 32 0 23 t i p value 0 38 0 20 0 23 0 19 0168539 5 8 3 65 0 45 1 10 2 37 9 20 48 27 27 12 32 11 37 10 10 79 17 27 4 96 14 22 11 30 79 24 77 32 35 30 09 12 17 14 19 77 28 49 26 54 13 15 56 9 32 19 12 14 45 14 1 59 0 681 1 47 0 951 15 0 181 5 45 Chapter 5 Experiments and Results 5 46 Table 5 5 AmpF STR Sinofiler kit allele frequencies continued African American aeara 20 p value 0 06 0 45 0 13 0 40 018 51 7 9 t t t 0 24 10 0 16 1 10 0 24 10 2 0 16 t t 11 0 48 1 14 0 74 0 71 12 5 40 5 91 14 52 9 00 12 2 t t 0 18 t 13 4 60 15 13 05 15 17 13 2 0 79 t t t 1
46. 16 capillaries run of the AmpF STR Sinofiler Allelic Ladder on the Applied Biosystems 3130x Genetic Analyzer 36 cm capillary and POP 4 polymer The internal size standard that was used was GeneScan 500 LIZ Size Standard The results were obtained within a set of injections on a single capillary array Sample alleles may occasionally size outside of the 0 5 nt window for a respective allelic ladder allele because of measurement error The frequency of such an occurrence is lowest in detection systems having the smallest standard deviations in sizing Figure 5 1 on page 5 4 illustrates the tight clustering of allele sizes obtained on the Applied Biosystems 3130x Genetic Analyzer where the standard deviation in sizing is typically less than 0 15 nt The instance of a sample allele sizing outside the 0 5 nt window because of measurement error is relatively rare when the standard deviation in sizing is approximately 0 15 nt or less Smith 1995 For sample alleles that do not size within a 0 5 nt window the PCR product must be rerun to distinguish between a true off ladder allele versus measurement error of a sample allele that corresponds with an allele in the allelic ladder Repeat analysis when necessary provides an added level of confidence to the final allele assignment The GeneMapper ID software v3 2 1 and later automatically flag sample alleles that do not size within the prescribed window around an allelic l
47. 218 87 0 076 0 101 19 222 5 222 92 0 067 0 099 20 226 53 226 95 0 069 0 102 21 230 57 231 0 084 0 094 22 234 61 235 07 0 067 0 103 23 238 65 239 11 0 074 0 102 24 242 69 243 16 0 073 0 114 25 246 74 247 23 0 078 0 119 26 250 79 251 29 0 079 0 109 26 2 252 8 253 32 0 07 0 121 27 254 8 255 32 0 072 0 114 28 258 85 259 38 0 075 0 118 29 262 92 263 46 0 081 0 117 30 267 01 267 56 0 081 0 119 30 2 268 84 269 39 0 08 0 13 31 2 272 91 273 48 0 081 0 13 AmpFSTR Sinofiler PCR Amplification Kit User Guide Chapter 5 Experiments and Results Table 5 1 Precision results of five runs 16 capillaries run of the AmpF STR Sinofiler Allelic Ladder continued Applied Biosystems 3130x Genetic Analyzer Allele Mean Standard Deviation 32 2 276 99 277 56 0 09 0 14 33 2 281 06 281 64 0 079 0 134 42 2 318 17 318 74 0 089 0 141 43 2 322 29 322 85 0 091 0 145 44 2 326 41 326 95 0 083 0 148 45 2 330 53 331 07 0 089 0 136 46 2 334 53 335 06 0 078 0 13 47 2 338 62 339 12 0 084 0 144 48 2 342 77 343 24 0 083 0 147 50 2 350 88 351 3 0 07 0 121 51 2 354 83 355 24 0 074 0 125 vWA 11 154 27 154 51 0 059 0 07 12 158 44 158 7 0 052 0 087 13 162 57 162 84 0 047 0 1 14 166 8 167 09 0 057 0 099 15 170 72 171 01 0 053 0 101 16 174 75 175 04 0 059 0 098 17 178 73 179 02 0 05 0 098 18 182 68 182 96 0 057 0 094 19 186 64 186 9 0 039 0 081 AmpF amp TR Sinofiler
48. 3 14 15 11 12 02 1338 2435 37 1 15 16 17 18 19 20 21 22 23 19 23 24 25 26 27 28 0195433 19412 13 1 9 10 11 12 12 2 13 13 2 14 NED 14 15 14 2 15 15 2 16 16 2 17 17 2 vWA 12p12 pter 11 12 13 14 15 16 17 18 19 17 18 20 21 22 23 24 012 391 12 13 2 14 15 16 17 18 19 19 3 20 18 20 21 22 23 24 25 26 27 018 51 18421 3 7 9 10 10 2 11 12 13 13 2 15 19 14 14 2 15 16 17 18 19 20 21 22 23 24 25 26 27 AmpF amp TR Sinofiler PCR Amplification Kit User Guide 1 3 Chapter1 Overview Table 1 1 AmpF STR Sinofiler PCR Amplification Kit loci and alleles continued Locus Chromosome Alleles Included in Sinofiler Dve Label Control Designation Location Allelic Ladder y DNA 9947A Amelogenin X p22 1 22 3 X Y X Y p11 2 D6S1043 6916 1 9 10 11 12 13 14 15 16 17 12 18 18 19 20 21 21 3 22 23 24 25 FGA 4q28 17 18 19 20 21 22 23 24 25 23 24 26 26 2 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 1 4 AmpFSTR Sinofiler PCR Amplification Kit User Guide Product Overview Allelic Ladder Figure 1 1 shows the allelic ladder for the AmpF STR Sinofiler kit See Allelic Ladder Requirements on page 3 2 for information on ensuring accurate genotyping 100 120 140 160 180 200 220 240 260 280 300 320 340 360 1200
49. 3100 Avant Genetic Analyzer User Guide Data Collection v1 0 4333549 ABI Prisv 3100 3100 Avant Genetic Analyzers Protocols for Processing AmpFSTR 4332345 PCR Amplification Kit PCR Products User Bulletin ABI 310 Genetic Analyzer User Guide Windows 4317588 Installation Procedures and New Features for GeneMapper ID Software v3 2 User 4352543 Bulletin GeneMapper ID Software Versions 3 1 and 3 2 Human Identification Analysis Tutorial 4335523 GeneMapper ID Software Version 3 1 Human Identification Analysis User Guide 4338775 Quantifiler Kits Quantifiler Human DNA Quantification Kit and Quantifiler Y Human 4344790 Male DNA Quantification Kit User s Manual GeneMapper ID Software v3 2 1 Patch User Bulletin 4382255 amp Identifiler PCR Amplification Kit User s Manual 4323291 AmpF amp TR Sinofiler PCR Amplification Kit User Guide xi Preface Note For additional documentation see How to Obtain Support on page xii Send Us Your Applied Biosystems welcomes your comments and suggestions for Comments improving its user documents You can e mail your comments to techpubs appliedbiosystems com IMPORTANT The e mail address above is only for submitting comments and suggestions relating to documentation To order documents download PDF files or for help with a technical question go to www appliedbiosystems com then click the link for Support See
50. 3130x Instrument for Electrophoresis 3 3 Preparing Samples for Electrophoresis on the 3100 3100 Avant 3130 3130x Instrument 3 4 Setting Up the 310 Instrument for Electrophoresis 3 6 Preparing Samples for Electrophoresis on the 310 Instrument 3 7 AmpF amp TR Sinofiler PCR Amplification Kit User Guide 3 1 Chapter 3 Performing Electrophoresis Allelic Ladder Requirements To accurately genotype samples you must run an allelic ladder sample along with the unknown samples For samples that are run on the ABI PRISM 310 Genetic Analyzer Run at least one allelic ladder for every 10 sample injections ABI PRISM 3100 or Applied Biosystems 3130 series instruments Run at least one allelic ladder per every set of 16 samples Applied Biosystems 3130x or ABI PRISM 3100 One ladder per injection one injection 16 samples 15 samples 1 allelic ladder Applied Biosystems 3130 or ABI PRISM Avant One ladder for every 4 injections one injection 4 samples IMPORTANT Variation in laboratory temperature can cause changes in fragment migration speed which can cause sizing variation Applied Biosystems recommends the above frequency of allelic ladder injections which should account for normal variation in run speed However during internal validation studies verify the required allelic ladder injection frequency
51. 4 6 67 18 18 16 36 13 03 14 2 0 32 t t 15 18 73 22 95 11 40 10 90 15 2 t t t 0 24 16 16 83 14 55 14 15 15 40 17 15 71 7 27 12 13 15 40 18 11 43 4 09 9 01 8 77 19 9 52 2 73 4 41 3 79 20 5 24 2 05 1 47 2 13 21 3 02 2 73 0 551 2 61 22 0 79 1 82 0 18 0 95 AmpF amp TR Sinofiler PCR Amplification Kit User Guide Population Data Table 5 5 AmpF STR Sinofiler kit allele frequencies continued AmpFSTR Sinofiler PCR Amplification Kit User Guide African American 020 23 0 16 0 68 0 55 0 95 24 t 0 45 t 0 24 25 t 0 181 t 26 t t t t 27 t t t 0 24 p value 0 37 0 55 0 39 0 41 019 433 9 0 231 10 0 95 0 241 11 8 57 0 181 1 18 11 2 0 161 t 0 24 12 11 43 3 86 8 64 7 11 12 1 0 181 t 12 2 4 60 0 911 1 18 13 26 67 32 05 26 10 23 22 13 2 4 92 3 64 1 10 5 69 14 20 79 21 14 33 64 25 83 14 2 4 92 12 05 2 76 5 69 15 6 51 5 45 16 91 14 45 15 2 4 76 16 14 2 21 9 24 16 1 75 1 36 6 62 3 08 16 2 3 33 2 73 1 10 2 13 5 47 Chapter 5 Experiments and Results 5 48 Table 5 5 AmpF STR Sinofiler kit allele frequencies continued African American aeara 020 17 t t 0 18 0 24 17 2 0 63 t 0 47 18 t 0 18 t 18 2 t t 0 18 t p value 0 32 0 09 0 00918 0 37 021511
52. 5 AmpFSTR Sinofiler PCR Amplification Kit User Guide 5 7 Chapter 5 Experiments and Results 5 8 Table 5 1 Precision results of five runs 16 capillaries run of the AmpF STR Sinofiler Allelic Ladder continued Applied Biosystems 3130x Genetic Analyzer Allele Mean Standard Deviation 8 264 25 264 65 0 065 0 093 9 268 24 268 66 0 065 0 093 10 272 23 272 65 0 068 0 082 11 276 24 276 65 0 064 0 085 12 280 25 280 67 0 063 0 103 13 284 26 284 68 0 062 0 093 14 288 28 288 7 0 063 0 089 15 292 29 292 72 0 058 0 095 018551 7 280 24 280 62 0 066 0 092 9 288 35 288 77 0 061 0 087 10 292 41 292 83 0 066 0 101 10 2 294 41 294 83 0 055 0 099 11 296 49 296 91 0 064 0 099 12 300 56 301 0 053 0 101 13 304 68 305 1 0 056 0 105 13 2 306 7 307 12 0 046 0 112 14 308 79 309 19 0 064 0 101 14 2 310 82 311 23 0 062 0 106 15 312 89 313 31 0 071 0 104 16 317 317 41 0 068 0 105 AmpFSTR Sinofiler PCR Amplification Kit User Guide Accuracy Precision and Reproducibility Table 5 1 Precision results of five runs 16 capillaries run of the AmpF STR Sinofiler Allelic Ladder continued Applied Biosystems 3130x Genetic Analyzer Allele Mean Standard Deviation 17 321 11 321 49 0 063 0 111 18 325 2 325 58 0 066 0 104 19 329 29 329 66 0 062 0 11 20 333 4 333
53. 5 56 Table 5 5 AmpF STR Sinofiler kit allele frequencies continued African American aeaa 20 13 1 27 t 0 37 t 14 7 14 24 09 8 27 6 87 15 20 48 2 73 12 87 13 74 16 23 97 17 27 20 04 27 73 17 20 32 23 64 25 92 27 73 18 15 87 19 77 20 04 16 11 19 6 83 10 68 10 11 6 16 20 2 70 1 36 2 39 1 18 21 0 48 t t s 22 t t t t 23 0 16 P 24 2 t p value 0 168 0 44 0 54 0 11 t Aminimum allele frequency 0 79 for the African American database 1 1496 for the Asian database 0 9296 for the U S Caucasian database and 1 1896 for the U S Hispanic database is suggested by the National Research Council in forensic calculations p value 0 05 for exact test for HWE Low Frequency Alleles Some alleles of the Sinofiler kit loci occur at a low frequency For these alleles a minimum frequency five divided by 2n where n equals the number of individuals in the database was assigned for the Sinofiler kit African American Asian U S Caucasian and U S Hispanic databases as suggested in the 1996 report of the Committee on DNA Forensic Science National Research Council 1996 These databases are summarized in Table 5 6 on page 5 59 The minimum reportable genotype frequency at each locus is 1 42 X 107 for the AmpF amp TR Sinofiler PCR Amplification Kit User Guide Population Data African American database 2 43 X 10 4 for the Asian
54. 52 0 068 0351358 12 111 45 111 62 0 047 0 071 13 115 54 115 73 0 051 0 072 14 119 53 119 7 0 044 0 08 15 123 44 123 6 0 056 0 076 16 127 63 127 8 0 047 0 078 17 131 83 132 01 0 042 0 076 18 135 93 136 11 0 047 0 068 19 140 03 140 22 0 045 0 067 055818 7 160 14 160 51 0 062 0 097 8 164 11 164 46 0 066 0 123 9 168 05 168 42 0 064 0 111 10 172 01 172 36 0 065 0 113 11 175 91 176 28 0 055 0 111 12 179 81 180 17 0 072 0 11 13 183 68 184 04 0 064 0 103 14 187 55 187 89 0 067 0 108 AmpF amp TR Sinofiler PCR Amplification Kit User Guide Accuracy Precision and Reproducibility Table 5 1 Precision results of five runs 16 capillaries run of the AmpF STR Sinofiler Allelic Ladder continued Applied Biosystems 3130x Genetic Analyzer Allele Mean Standard Deviation 15 191 39 191 73 0 065 0 08 16 195 23 195 55 0 071 0 084 D6S1043 9 126 37 126 61 0 069 0 096 10 130 22 130 47 0 064 0 091 11 134 13 134 38 0 071 0 084 12 138 08 138 33 0 065 0 088 13 142 26 142 55 0 058 0 087 14 146 48 146 79 0 062 0 098 15 150 8 151 12 0 068 0 101 16 154 98 155 34 0 064 0 102 17 159 04 159 44 0 074 0 117 18 163 01 163 42 0 061 0 134 19 166 96 167 36 0 066 0 132 20 170 87 171 28 0 081 0 143 21 174 78 175 19 0 075 0 121 21 3 177 66 178 07 0 073 0 12 22 178 67 179 09 0 078 0 121 23 182 61 183 03 0 082 0 116 24 186 48 186 9 0 074 0 125 25 190 42 190 83 0 077 0 105
55. 5_Sinofiler_GS500 size standard is provided as a default size standard in GeneMapper JD software v3 3 To import an HID Size Standard 1 Select Tools GeneMapper Manager to open the GeneMapper Manager 2 Import a size standard a Select the Size Standards tab then click Import 7 GeneMapper Manager Projects Analysis Methods Table Settings Plot Settings Matrices Size Standards Last Saved Owner Type Description 377 G5 HID GS500 2005 09 20 14 10 2 gmid Basic Advanced Factory Provided 377 F HID GS500 2006 09 20 14 10 2 gmid Basic Advanced Factory Provided CE G5 HID GS500 2006 09 20 14 10 2 gmid Basic Advanced Factory Provided CE F HID GS500 2006 09 20 14 10 2 Basic Advanced Provided b Navigate to then open the GMID Sinofiler files folder 3 Select CE G5 Sinofiler GS500 then click Import to import the Sinofiler HID v1 analysis method into the GeneMapper JD database Import Size Standard Method Look in le GMID Sinofiler files c cE_G5_Sinofiler_G Ce Sinofiler v1 File name CE_G5_Sinofiler_GS500 xml Desktop Files of type Files AmpF amp TR Sinofiler PCR Amplification Kit User Guide Analyzing and Editing Sample Files with GeneMapper ID Software Analyzing and Editing Sample Files with GeneMapper ID Software Analyzing analyze a project a P
56. 60 values 5 59 Index 3 quality flags settings 4 15 Quantifiler kit description 2 5 quantifying DNA methods 2 4 R radioactive waste handling ix reaction volume final for PCR 2 7 reactions preparing for PCR 2 6 reagents low TE buffer 2 3 not included with kit 1 10 run module electrophoresis 3 3 3 6 S safety biological hazards x chemical waste viii guidelines vii ix size deviation sample alleles and ladder alleles 5 4 size standard GeneMapper ID software 4 17 sizing precision 5 4 species specificity 5 30 split peaks A nucleotide addition 5 24 figure 5 25 stability DNA 5 34 standards for samples 1 10 STRBase 5 43 stutter percent D198433 0125391 and D18S51 loci 5 21 0381358 055818 0135317 2165539 0251338 loci 5 20 055818 and FGA 5 22 0851179 0251338 075820 and CSFIPO loci 5 19 stutter products 5 18 Index 4 Technical Support contacting xii thermal cyclers for use with kit 2 2 programming 2 8 training information xii troubleshooting causes and actions 2 U user attention words described V validation characterization ofloci 5 28 developmental 5 2 effect of DNA quantity 5 32 experiments to evaluate 5 2 importance of 5 2 importance of DNA quantitation 5 32 mixture studies 5 37 mutation rate 5 58 population data 5 42 probability of identity 5 59 probability of paternity exclusion 5 61 sensitivity 5 32 size d
57. 60 270 280 290 300 310 320 330 340 350 360 800 90 1 1 0 100 110 420 130 140 150 160 170 180 130 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350 360 2000 0 1 9 Figure 5 12 Amplification of DNA mixtures at various ratios Limit of Detection of the Minor Component Mixtures of two DNA samples were examined at various ratios 0 1 1 1 3 1 7 1 15 1 1 0 The total amount of genomic input DNA mixed at each ratio was 1 ng The samples were amplified in a GeneAmp PCR System 9700 then electrophoresed and detected using an Applied Biosystems 3130x Genetic analyzer The results of the mixed DNA samples are shown in Figure 5 12 where samples A and B were mixed according to the indicated ratios The minor component allele calls at non overlapping loci are highlighted The amplification of the minor contributor at 3 1 and 7 1 0 875 0 125 ng mixture ratios was readily typeable 15 1 ratios generally resulted in partial profiles for the minor component 5 40 AmpF amp TR Sinofiler PCR Amplification Kit User Guide Mixture Studies Table 5 4 shows the profiles of the samples in Figure 5 12 on page 5 40 Table 5 4 Genotypes of mixed DNA samples Allele Cent DNA DU Profile Sample B D8S1179 12 13 14 15 021 11 28 31 28 30 D7S820 7 12 8 9 CSF1PO 11 12 10 0351358 15 16 15 18 055818 11 8 1
58. A is severely degraded If possible evaluate the quality of DNA sample by running an agarose gel If DNA is degraded reamplify with an increased amount of DNA Dilution of test sample DNA or wrong buffer e g wrong EDTA concentration Redilute DNA using TE Buffer with 0 1 mM EDTA More than one allele present at a locus Presence of exogenous DNA Use appropriate techniques to avoid introducing foreign DNA during laboratory handling Too much DNA in reaction Use recommended amount of template DNA 0 5 to 1 25 ng Mixed sample Amplification of stutter product n 4 nt position See Stutter Products on page 5 18 Incomplete 3 A base addition n 1 nt position See Addition of A Nucleotide on page 5 23 Be sure to include the final extension step of 60 C for 60 min in the PCR Signal exceeds dynamic range of instrument off scale data Quantitate DNA and reamplify sample adding 0 5 to 1 25 ng of DNA Poor spectral separation bad matrix Follow the steps for creating a matrix file Confirm that Filter Set G5 modules are installed and used for analysis A 4 AmpF STR Sinofiler PCR Amplification Kit User Guide Table A 1 Troubleshooting continued Troubleshooting Observation Possible Causes Recommended Actions Some but not all loci visible on electropherogram Test sample DNA is severely degr
59. AmpF STR Sinofiler PCR Amplification Kit User Guide Applied Biosystems AmpF STR Sinofiler PCR Amplification Kit User Guide Copyright 2012 Life Technologies Corporation All rights reserved 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 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 Applied Biosystems AB Design ABI PRISM AMPFLSTR GeneAmp GeneMapper GeneScan Identifiler LIZ PET Profiler Plus Quantifiler SGM Plus and VIC are registered trademarks and FAM GeneScan Hi Di MicroAmp NED POP 4 and Sinofiler are trademarks of Applied Biosystems or its subsidiaries in the U S and or certain other countries AmpliTaq AmpliTaq Gold and TaqMan are registered trademarks of Roche Molecular Systems Inc Windows NT is a registered trademark of Microsoft Corporation All other trademarks are the sole property of their respective owners Part Number 4384256 Rev E
60. Collection Run Module References Software Windows XP 3 11 GS STR POP4 1 mL G5 2 5 ABI PRISM 310 Genetic Analyzer User s Manual Windows PN 4317588 ABI 310 Protocols for Processing AmpF4STR PCR Amplification Kit Products with Microsoft Windows NT Operating System User Bulletin PN 4341742 and Windows 2000 Windows 3 0 GS STR POP4 1 mL G5 2 5 ABI PRISM 310 Genetic Analyzer User s Manual Windows PN 4317588 ABI 310 Protocols for Processing AmpF4STR PCR Amplification Kit Products with Microsoft Windows NT Operating System User Bulletin PN 4341742 t Applied Biosystems conducted concordance studies for the Sinofiler kit using this configuration 3 6 AmpFSTR Sinofiler PCR Amplification Kit User Guide Preparing Samples for Electrophoresis on the 310 Instrument Preparing Samples for Electrophoresis on the 310 Instrument Preparing the Prepare the samples for electrophoresis on the 310 instrument Samples immediately before loading To prepare samples for electrophoresis 1 Calculate the volume of Hi Di Formamide and GeneScan 500 LIZ Internal Size Standard needed to prepare the samples using the table below Volume Reagent Per Reaction uL GeneScan 500 LIZ Size Standard 0 5 Hi Di Formamide 24 5 Note Include additional samples in your calculations to provide excess volume f
61. F STR PCR Reaction Mix AmpliTaq Gold DNA Polymerase and AmpF STR Sinofiler Primer Set reagents IMPORTANT The fluorescent dyes attached to the primers are light sensitive Protect the AmpF STR Sinofiler Primer Set from light when not in use Also protect the AmpF STR Sinofiler Allelic Ladder GeneScan 500 LIZ Size Standard and amplified fluorescently labeled PCR products from light To prepare the master mix Determine the total number of samples including controls 2 IMPORTANT Vortex the following reagents for 5 sec AmpF STR PCR Reaction Mix AmpliTaq Gold DNA Polymerase AmpF STR Sinofiler Primer Set CHEMICAL HAZARD AmpliTaq Gold DNA Polymerase may cause eye and skin irritation It may cause discomfort if swallowed or inhaled Read the MSDS and follow the handling instructions Wear appropriate protective eye wear clothing and gloves 3 Spin the tubes briefly a microcentrifuge to remove any liquid from the caps 4 Select a clean unused tube for the master mix If you are preparing Then use a 34 samples and controls 1 5 mL microcentrifuge tube 85 110 samples and controls 2 0 mL microcentrifuge tube gt 110 samples and controls Tube that is appropriate AmpFSTR Sinofiler PCR Amplification Kit User Guide Preparing the Reactions To prepare the master mix continued 5 Calculate the requir
62. Genetic Analyzer using another AmpF STR kit Lack of complete nucleotide addition may be observed in Sinofiler kit results when the amount of input DNA is greater than the recommended protocols because more time is needed for AmpliTaq Gold DNA Polymerase to add the nucleotide to all molecules as more PCR product is generated Amplification of too much input DNA may also result in off scale data AmpF amp TR Sinofiler PCR Amplification Kit User Guide 5 25 Chapter 5 Experiments and Results 5 26 Artifacts Artifacts and anomalies are seen in all molecular biological systems Artifacts are typically reproducible and anomalies are nonreproducible intermittent occurrences that are not consistently observed in a system for example spikes and baseline noise Artifacts have been seen in data produced on genetic analyzers when using the Sinofiler kit In amplified samples artifacts in the non calling region may appear in the blue 95 100 nt dye Low level artifacts in the calling region may appear in the blue 118 nt green 97 120 and 189 nt and black 95 100 164 nt dyes depending on the sensitivity of the instrument Figure 5 7 on page 5 27 shows examples of baseline noise and artifacts in an electropherogram while using the Sinofiler kit Genotyping may result in the detection of these artifacts as off ladder alleles or OL Alleles You should consider possible noise and artifacts when interpreting data Am
63. IMPORTANT If you close the Panel Manager without clicking OK the panels and bins are not imported into the GeneMapper ID database 4 8 AmpFSTR Sinofiler PCR Amplification Kit User Guide Setting Up GeneMapper ID Software for Analyzing AnpFSTR Sinofiler Kit Data Importing an HID analysis method for the AmpF STR Sinofiler PCR Analysis Method Amplification Kit uses the HID Advanced Mode Peak Detection v3 2 1 and v3 3 Algorithm This analysis method provides users with the same analysis parameters available in GeneScan Software v3 7 1 for the Windows operating system Note The HID Advanced Mode analysis method below makes use of the AmpFLSTR Sinofiler Bins vl file described in Table 4 1 on page 4 12 Use the following procedure to import the analysis method for the Sinofiler kit from the folder that you downloaded from the Applied Biosystems web site into the GeneMapper ID software database Refer to step 1 on page 4 4 for downloading instructions Note Sinofiler v1 33 analysis method is provided as a default analysis method in GeneMapper D software v3 3 By following the above procedure it is possible to manually import additional analysis methods besides the one supplied with GeneMapper software v3 3 To import the HID Advanced Mode analysis method into GeneMapper D software 1 Select Tools GeneMapper Manager to open the GeneMapper Manager 2 Import an analysis met
64. adder allele Although the precision within a gel or set of capillary injections is very good the determined allele sizes vary between platforms Cross platform sizing differences occur from a number of factors including type and concentration of polymer mixture run temperature and electrophoresis conditions Variations in sizing can also occur between runs on the same instrument and between runs on different instruments because of these factors Applied Biosystems strongly recommends that the allele sizes obtained be compared to the sizes obtained for known alleles in the AmpF STR Sinofiler Allelic Ladder from the same run and then be converted to genotypes as described in Before You Start on AmpF amp TR Sinofiler PCR Amplification Kit User Guide 5 5 Chapter 5 Experiments and Results 5 6 page 4 2 Refer to Table 5 1 for the results of five runs of the AmpF STR Sinofiler Allelic Ladder For more information on precision and genotyping see Lazaruk et al 1998 and Mansfield et al 1998 In Table 5 1 the mean size for all the alleles in each run 16 capillaries was calculated The mean range shown in the table is the lowest and highest mean size values of the five runs Similarly the standard deviation for the allele sizing was calculated for all the alleles in each run The standard deviation range shown in Table 5 1 is the lowest and highest standard deviation values of the five runs Table 5 1 Precision r
65. aded If possible evaluate the quality of DNA sample by running an agarose gel If DNA is degraded reamplify with an increased amount of DNA Test sample contains high concentrations of a PCR inhibitor for example heme compounds certain dyes AmpFSTR Sinofiler PCR Amplification Kit User Guide Quantitate DNA then add minimum necessary volume Repeat test Wash the sample Centricon 100 A 5 Appendix Troubleshooting A 6 amp Sinofiler PCR Amplification Kit User 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 Forensic Sci 39 362 372 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 Barber M D and Parkin B H 1996 Sequence analysis and allelic designation of the two short tandem repeat loci D18S51 and 1851179 Intl J Legal Med 109 62 65 Baron H Fung S Aydin A Bahrig S Luft Schuster 1996 Oligonucleotide ligation assay OLA 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
66. ana 5 37 Population 5 42 Mutation Rate 12 ev EE b a WS 5 58 Probability 1 5 59 Probability of Paternity Exclusion 5 61 AmpF amp TR Sinofiler PCR Amplification Kit User Guide 5 1 Chapter 5 Experiments and Results Overview Experiments Using AmpF STR Sinofiler PCR Amplification Kit 5 2 Importance of Validation Experiment Conditions This chapter provides results of the developmental validation experiments performed by Applied Biosystems using the AmpF STR Sinofiler PCR Amplification Kit Sinofiler kit Validation of a DNA typing procedure for human identification applications is an evaluation of the procedure s efficiency reliability and performance characteristics By challenging the procedure with samples commonly encountered in forensic and parentage laboratories the validation process uncovers attributes and limitations which are critical for sound data interpretation in casework Sparkes Kimpton Watson et al 1996 Sparkes Kimpton Gilbard et al 1996 Wallin et al 1998 Experiments to evaluate the performance of AmpF STR PCR Amplification Kit were performed at Applied Biosystems The experiments were performed according to the DNA Advisory Board DAB Quality Assurance Standards effective October 1 1998 DNA Advisory B
67. at 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 AmpFLSTR_Sinofiler_Panels_v1 file Note For more information about allele filters refer to the GeneMapper ID Software Version 3 1 Human Identification Analysis User Guide Chapter 3 PN 4338775 and the Installation Procedures and New Features for GeneMapper ID Software v3 2 User Bulletin PN 4352543 4 12 AmpF amp TR Sinofiler PCR Amplification Kit User Guide Setting Up GeneMapper ID Software for Analyzing AnpFSTR Sinofiler Kit Data Table 4 1 Sinofiler_HID_v1 Advanced Mode analysis method settings continued Tab Settings Peak Detector IMPORTANT Laboratories need to perform the appropriate internal validation studies to determine the peak amplitude threshold highlighted in red below that allows for reliable interpretation of Sinofiler data Analysis Method Editor HID General Allele Peak Detection Algorithm amp dvanced v Ranges Peak Detection Analysis Sizing Peak Amplitude Thresholds Partial Range Partial Sizes 50 Start 2500 Start 5 75 d Pe 50 0 50 Stop 10000 Stop Size 450 a Smoothing and Baselining Min Peak Half Smoothing O None Light O Heavy Peak Window Size Baseline Window
68. ata Table 4 1 Sinofiler_HID_v1 Advanced Mode analysis method settings Tab Settings General Name Sinofiler_HID_v1 Analysis Method Editor HID Allele Peak Detector Peak Quality Quality Flags r Analysis Method Description Name Sinofiler_HID_v1 Description Default Sinofiler HID analysis method version 1 Instrument Analysis Type HID AmpF amp TR Sinofiler PCR Amplification Kit User Guide 4 11 Chapter 4 Analyzing Data Table 4 1 Sinofiler HID v1 Advanced Mode analysis method settings continued Tab Settings Allele Analysis Method Editor HID General Peak Detector Peak Quality Quality Flags Bin Set AmpFLSTR_Sinofiler_Bins_v1 Use marker specific stutter ratio if available Marker Repeat Type Tri Tetra Cut off Value 00 oo o oo MinusA Ratio 00 oo 00 MinusA Distance joo joo o oo 00 00 lop Minus Stutter Ratio 00 00 Minus Stutter Distance 00 oo 00 00 oo oo Plus Stutter Ratio 00 Plus Stutter Distance From 00 0 0 00 To 00 0 oo Amelogenin Cutoff 0 0 Range Filter Factory Defaults e GeneMapper ID Software v3 2 1 and v3 3 allow you to specify four types of marker repeat motifs tri tetra penta and hexa You can enter parameter values for each type of repe
69. ation results in 107 nt and 113 nt products from the X and Y chromosomes respectively Sizes are the actual nucleotide size according to sequencing results including 3 A nucleotide addition The remaining Sinofiler kit loci are all tetranucleotide short tandem repeat STR loci The length differences among alleles of a particular locus result from differences in the number of 4 nt repeat units The loci D6S1043 and D128391 amplified by the Sinofiler kit are compound STR markers The D6S1043 alleles contain repeat unit sequences ATCT and ATGT that can differ in number and relative position within the repeat region The most common repeat motif ATCT is referred to as the core repeat sequence The D128391 locus consists of repetitive basic structures of AGAT AGAC AGAT Sequence variations within the repeat region and allele distributions in these two loci have been reported in various populations Chen et al 1999 Chen et al 2004 Glock et al 1997 Hu et al 2004 Junge and Madea 1998 Klintschar et al 1998 Li et al 2004 Liu et al 2005 Lu et al 2003 Shin et al 2004 Su et al 2004 Waiyawuth et al 1998 Wu et al 2004 Yu et al 2003 All the alleles in the AmpF STR Sinofiler Allelic Ladder have been been subjected to sequencing at Applied Biosystems In addition other groups in the scientific community have sequenced alleles at some of these loci Among the various sources of sequence data on t
70. ation tube purified and amplified without transferring the evidence Applied Biosystems studies have indicated that a 1 2 mm bloodstained punch contains approximately 5 20 ng DNA Accordingly an appropriate cycle number for this high quantity of DNA is 25 cycles It is recommended that each laboratory determine the cycle number based on individual validation studies In the example shown in Figure 2 1 a 1 2 mm punch of a bloodstained FTA card was purified using three washes with FTA Purification Reagent and two washes with 1X TE buffer After drying at room temperature overnight the punch was then amplified directly in the MicroAmp tube for 25 cycles 100 110 420 130 440 450 160 470 180 190 200 240 220 230 240 250 260 270 290 290 300 310 320 330 340 14004 m 1000 soo 6004 mol 200 0 Figure 2 1 AmpF STR Sinofiler PCR Amplification Kit results from a 1 2 mm FTA bloodstain punch 25 cycle amplification analyzed on the ABI PRISM 3130x Genetic Analyzer AmpF amp TR Sinofiler PCR Amplification Kit User Guide 2 9 Chapter 2 Amplification 2 10 AmpF amp TR Sinofiler PCR Amplification Kit User Guide Part Number 4384256 Rev 03 2012 Chapter 3 Electrophoresis AmpFSTR Sinofiler PCR Amplification Kit User Guide Performing Electrophoresis This chapter covers Allelic Ladder Requirements 3 2 Setting Up the 3100 3100 Avant or 3130
71. be decapper autoclavable Vortex Amplified DNA IMPORTANT The following GeneAmp PCR Systems should never Work Area leave the Amplified DNA Work Area Silver 96 Well GeneAmp PCR System 9700 Gold plated silver block GeneAmp PCR System 9700 GeneAmp PCR System 9600 2 2 AmpFSTR Sinofiler PCR Amplification Kit User Guide Required User Supplied Materials and Reagents Required User Supplied Materials and Reagents Kit Contents and Each AmpF STR Sinofiler PCR Amplification Kit contains Storage materials sufficient to perform 200 reactions at a 25 1 reaction volume See Kit Contents on page 1 9 for details on Sinofiler kit contents 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 Sinofiler Allelic Ladder and GeneScan 500 LIZ Size Standard should also be protected from light Minimize freeze thaw cycles User Supplied In addition to the Sinofiler kit reagents the use of low TE buffer Reagents 10 mM Tris 0 1 mM EDTA pH 8 0 is recommended You prepare the buffer as described in the following table or order it from Teknova Cat T0223 To prepare low TE buffer 1 Mix together 10 mL of 1 M Tris HCl pH 8 0 0 2 mL of 0 5 M EDTA pH 8 0 990 mL glass distilled or deionized water CHEMICAL HAZARD EDTA Exposure causes eye irritation Read the MSDS and follow the hand
72. d 1 3 master mix 1 9 primers 1 2 1 9 purpose 1 2 reagents 1 9 thermal cyclers for use with 2 2 kit performance comparison DNase I figure 5 35 hematin figure 5 36 L LIZ size standard about 1 10 volume per reaction 3 4 3 7 loci amplified 1 3 characterization 5 28 chromosomal location 1 3 combined genotype frequency 5 57 dye label 1 3 genotype frequency in population 5 56 mapping 5 29 low TE buffer preparation 2 3 M marker displaying Bin view of 4 8 materials and equipment 1 9 materials not included with kit 1 10 menu commands conventions for describing mixed samples resolution of genotypes 5 39 mixture studies 5 37 MSDSs description vii multicomponent analysis 1 7 AmpF amp TR Sinofiler PCR Amplification Kit User Guide mutation rate 5 58 mutation studies 5 58 mutation STR 5 58 N navigation pane displaying list of panels 4 7 Panel Manager 4 5 O off ladder alleles 4 3 5 4 operating systems 1 7 3 3 3 6 Panel Manager 4 4 panels viewing 4 7 PCR performing 2 8 setup 2 2 PCR inhibitor hematin 5 35 PCR work areas 2 2 peak detection parameters 4 13 peak height ratios table of alleles 5 37 peak height minimum 4 13 percent stutter highest value for locus 5 18 off scale peaks 5 19 relation to allele length 5 18 PQV thresholds 4 15 precision and size windows 5 4 precision sizing 5 4 primers about 1 2 Amelogenin 5 28 probability of identity definition 5 60 populations 5
73. d percent stutter Multicomponent analysis of off scale data is not accurate This inaccuracy results in poor spectral separation pull up Incomplete nucleotide addition The sample can be reamplified using less DNA When the total number of allele copies added to the PCR is extremely low unbalanced amplification of the alleles may occur because of stochastic fluctuation 5 32 AmpF amp TR Sinofiler PCR Amplification Kit User Guide Sensitivity Individual laboratories may find it useful to determine an appropriate minimum peak height threshold based on their own results and instruments using low amounts of input DNA 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350 RE e e e E A 3 AK 160 170 180 190 200 210 20 230 240 250 260 270 280 290 300 310 320 330 340 350 E M M MM 4 160 170 180 190 200 210 220 230 240 250 260 270 280 230 300 310 320 330 340 350 0 25 ng 160 170 180 130 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350 woy 4004 5555555 d 160 170 480 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350 E E A E E A tt ttt A E A EE A tt E E E E M 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350 egative control Figure 5 9 Effect of amplifying varying amounts of of control DNA 9947A and negative control Note that the y axis scale is magnified for
74. e 50 mL Falcon MLS Tube decapper autoclavable MLS Deionized water PCR grade MLS Tris HCL pH 8 0 MLS 0 5 M EDTA MLS Vortex MLS t For the Material Safety Data Sheet MSDS of any chemical not distributed by Applied Biosystems contact the chemical manufacturer Before handling any chemicals refer to the MSDS provided by the manufacturer and observe all relevant precautions 1 14 AmpF amp TR Sinofiler PCR Amplification Kit User Guide Chapter 2 PCR Amplification AmpFSTR Sinofiler PCR Amplification Kit User Guide PCR Amplification This chapter covers PCR Work Rae oes Ra 2 2 Required User Supplied Materials and Reagents 2 3 Quantifying DNA 2 4 Preparing the 2 6 Performing 2 8 Amplification Using Bloodstained FTA Cards 2 9 AmpF amp TR Sinofiler PCR Amplification Kit User Guide 2 1 Chapter 2 PCR Amplification PCR Work Areas PCR Setup Work IMPORTANT The following items should never leave the PCR Setup Area Work Area Calculator Gloves disposable Marker pen permanent Microcentrifuge Microcentrifuge tubes 1 5 mL or 2 0 mL or other appropriate clean tube for Master Mix preparation Microcentrifuge tube rack Pipette tips sterile disposable hydrophobic filter plugged Pipettors Tu
75. ed amount of components as shown Note The formulation in the list below provides a slight overfill to allow for volume lost in pipetting Number of samples X 10 5 uL of AmpF STR PCR Reaction Mix Number of samples X 0 5 uL of AmpliTaq Gold DNA Polymerase Number of samples X 5 5 uL of AmpF STR Sinofiler Primer Set 6 Dispense the appropriate volume of each of the components from step 5 into a microcentrifuge tube 7 Vortex the master mix at medium speed for 3 sec then centrifuge the tube or plate briefly before opening the tubes 8 Dispense 15 of master mix into each reaction tube or plate well Preparing d Prepare the DNA samples Sinofiler Kit be RR Reactions DNA Sample To Prepare Negative Control Add 10 uL of low TE buffer to the reaction tube or plate well Your Sample Dilute a portion of your DNA sample with low TE buffer so that 0 50 1 25 ng of total DNA is in a final volume of 10 Add your sample to the reaction tube or plate well Positive Control Add 10 uL of control DNA 9947A 0 1 ng uL to the reaction tube or plate well Note The final reaction volume should be 25 2 Centrifuge the plate at 3 000 rpm for about 20 seconds in a tabletop centrifuge with plate holders to remove bubbles 3 Amplify the DNA in a GeneAmp PCR System 9600 or a Silver 96 Well GeneAmp PCR System 9700 or a Gold plated silver block GeneAmp PCR Syste
76. es 0 5 mL 401957 1 12 AmpF amp TR Sinofiler PCR Amplification Kit User Guide Table 1 3 User supplied materials continued Materials and Equipment Item Source Septa for 0 5 mL Sample Tubes 401956 DS 33 Matrix Standard Set 6FAM VIC9 NED PETS and 1179 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 MicroAmp 96 Well Full Plate Cover N8010550 MicroAmp 96 Well Tray Retainer Set 403081 POP 4 Polymer for the 310 Genetic Analyzer 402838 ABI 310 Genetic Analyzer User Guide PN 4317588 For a complete list of parts and accessories for the 310 instrument refer to Appendix B of the 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 Caps Strip N8010535 MicroAmp 96 Well Tray Retainer Set 403081 MicroAmp 96 Well Base N8010531 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 AmpFSTR Sinofiler PCR Amplification Kit User Guide Chapter 1 Overview Table 1 3 User supplied materials continued Item Source Tub
77. esults of five runs 16 capillaries run of the AmpF STR Sinofiler Allelic Ladder Applied Biosystems 3130x Genetic Analyzer Allele Mean Standard Deviation Amelogenin X 108 2 108 38 0 056 0 076 Y 114 33 114 52 0 047 0 073 CSF1PO 6 304 11 304 52 0 054 0 083 7 308 15 308 56 0 057 0 084 8 312 21 312 6 0 042 0 075 9 316 26 316 64 0 041 0 073 10 320 3 320 66 0 055 0 0768 11 324 34 324 69 0 051 0 077 12 328 37 328 71 0 035 0 078 13 332 42 332 72 0 045 0 072 14 336 45 336 74 0 04 0 073 15 340 47 340 74 0 037 0 081 AmpFSTR Sinofiler PCR Amplification Kit User Guide Accuracy Precision and Reproducibility Table 5 1 Precision results of five runs 16 capillaries run of the AmpF STR Sinofiler Allelic Ladder continued Applied Biosystems 3130x Genetic Analyzer Allele Mean Standard Deviation 012 391 8 215 54 215 79 0 05 0 066 9 219 5 219 77 0 054 0 071 10 223 5 223 79 0 047 0 067 11 227 42 227 71 0 045 0 062 12 231 38 231 68 0 046 0 066 13 235 33 235 63 0 042 0 067 14 238 35 238 67 0 053 0 075 15 239 3 239 63 0 054 0 07 0135317 8 216 67 217 02 0 06 0 07 9 220 64 221 01 0 07 0 087 10 224 64 225 01 0 07 0 095 11 228 63 229 01 0 058 0 087 12 232 7 233 11 0 05 0 08 13 236 6 237 0 074 0 087 14 240 54 240 95 0 067 0 088 15 244 53 244 97 0 063 0 093 0165539 5 252 29 252 66 0 068 0 08
78. eviation sample and ladder alleles 5 4 species specificity 5 30 stability 5 34 W WARNING description vi waste disposal guidelines 1 work area amplified DNA 2 2 PCR setup 2 2 workflow overview 1 6 AmpFSTR Sinofiler PCR Amplification Kit User Guide Worldwide Sales and Support Applied Biosystems vast distribution and service network composed of highly trained support and applications personnel reaches 150 countries on six continents For sales office locations and technical support please call our local office or refer to our Web site at www appliedbiosystems com Applied Biosystems is committed to providing the world s leading technology and information for life scientists Headquarters 850 Lincoln Centre Drive Foster City CA 94404 USA Phone 1 650 638 5800 Toll Free In North America 1 800 345 5224 Fax 1 650 638 5884 03 2012 Applied Biosystems Part Number 4384256 Rev
79. he Sinofiler kit loci there is consensus on the repeat patterns and structure of the STRs The Centre d Etude du Polymorphisme Humain CEPH has collected DNA from families of Utah Mormon French Venezuelan and Amish descent These DNA sets have been extensively studied all over the world and are routinely used to characterize the mode of AmpF amp TR Sinofiler PCR Amplification Kit User Guide Characterization of Loci inheritance of various DNA loci Each family set contains three generations generally including four grandparents two parents and several offspring Consequently the CEPH family DNA sets are ideal for studying inheritance patterns Begovich et al 1992 Three CEPH family DNA sets were examined ng of DNA from each sample was amplified using the AmpF STR Sinofiler kit followed by analysis using an Applied Biosystems 3130x Genetic Analyzer The families examined included 1333 9 offspring 1340 7 offspring and 1345 7 offspring representing 23 meiotic divisions In family 1340 we observed two parent offspring pairs with mutations at locus D8S1179 In family 1333 one mutation was identified at locus D128391 The genotypes differed by one repeat unit between the two generations These samples were reamplified using the AmpF STR Sinofiler and AmpF STR Identifiler kits to confirm the allele calls Calculation of a mutation rate based on these data would be inaccurate due to the small sample size
80. height of the stutter peak by the height of the main allele peak Peak heights were measured for amplified samples 840 at the loci used in the AmpF STR Sinofiler PCR Amplification Kit All data were generated on the Applied Biosystems 3130x Genetic Analyzer Some conclusions from these measurements and observations are For each Sinofiler kit locus the percent stutter generally increases with allele length as shown in Figure 5 2 to Figure 5 5 on pages 5 19 through 5 22 Smaller alleles display a lower level of stutter relative to the longer alleles within each locus Each allele within a locus displays a percent stutter that is consistent The highest observed percent stutter for each locus is included as the filtering step in GeneMapper D software v3 2 1 and later These values are shown in Table 5 2 on page 5 23 Peaks in the stutter position that are above the highest observed percent stutter will not be filtered Peaks in the stutter position that have not been filtered and remain labeled can be further evaluated For evaluation of mixed samples see Mixture Studies on page 5 37 AmpF amp TR Sinofiler PCR Amplification Kit User Guide Extra Peaks in the Electropherogram The measurement of percent stutter for peaks that are off scale 17 0 16 0 15 0 14 0 13 0 12 0 11 0 10 0 9 0 8 0 Percent Stutter 7 0 6 0 5 0 4 0 3 0 2 0 0 0 may be unusually high
81. hod for Advanced a Select the Analysis Methods tab then click Import GeneMapper Manager Projects Analysis Methods Table Settings Plot Settings Matrices Size Standards Name Last Saved owner Instrument Analysis Type Description SNePshot Defaut 2005 03 16 16 00 3 amid SNePshot Factory Provid YFiler 2005 03 28 08 29 4 grid 3100 lt gt b Navigate to then open the GMID_Sinofiler_files folder AmpF amp TR Sinofiler PCR Amplification Kit User Guide 4 9 Chapter 4 Analyzing Data 4 10 To import the HID Advanced Mode analysis method into GeneMapper D software continued 3 Select Sinofiler_HID_v1 then click Import to import the Sinofiler_HID_v1 analysis method into the GeneMapper database 79 Import Analysis Method Look in la GMID Sinofiler files 5 G5 05500 Sinofiler_HID_v1 File name Sinofiler HID v1 xml Desktop Files of type xML Files xml To view the settings in the Sinofiler_HID_v1 analysis method a Select the Analysis Methods tab b Select Sinofiler HID v1 in the Name column then click Open Table 4 1 on page 4 11 shows the settings for each tab of the Analysis Method Editor HID AmpF amp TR Sinofiler PCR Amplification Kit User Guide Setting Up GeneMapper ID Software for Analyzing AnpFSTR Sinofiler Kit D
82. ic tandem repeats Am J Hum Genet 49 746 756 AmpF amp TR Sinofiler PCR Amplification Kit User Guide Edwards A Hammond H A Lin J Caskey C T and Chakraborty R 1992 Genetic variation at five trimeric and tetrameric tandem repeat loci in four human population groups Genomics 12 241 253 Frank W Llewellyn B Fish et al 2001 Validation of the AmpF STR Profiler Plus PCR Amplification Kit for use in forensic casework Forensic 46 642 646 Glock B Dauber E M Schwartz D W Mayr W R 1997 Additional variability at the D128391 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 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 Hammond Jin L Zhong Caskey C and Chakraborty 1994 Evaluation of 13 short tande
83. ication Kit As the DNA became increasingly degraded the larger size loci became undetectable Figure 5 10 on page 5 35 AmpF amp TR Sinofiler PCR Amplification Kit User Guide Stability 1 DNA 160 170 180 190 200 210 220 230 2 0 250 260 270 280 290 300 310 320 330 340 350 0 Units DNasel 2 Units DNasel soot 400 200 _ ttt 3 Units DNasel 4 Units 1000 DNasel 600 400 0 12007 4 4 4 4000 5 Units 90 DNasel Effect of Inhibitors Hematin AmpF amp TR Sinofiler PCR Amplification Kit User Guide 6 Units DNasel Figure 5 10 Amplification of 1 ng Raji DNA samples sonicated and then treated with 0 2 3 4 5 and 6 units of DNase I Heme compounds have been identified as PCR inhibitors in DNA samples extracted from bloodstains DeFranchis 1988 Akane et al 1994 It is believed that the inhibitor is co extracted and co purified with the DNA and subsequently interferes with PCR by inhibiting polymerase activity To examine the effects of hematin on the amplification results obtained by the Sinofiler kit male DNA 007 1 ng input was amplified with increasing concentrations of hematin 0 uM 40 uM 50 uM 60 uM and 70 uM Figure 5 11 on page 5 36 No preferential amplification was observed in the presence of increasing amoun
84. leles EREE 851179 bue 1130 1785 13 021511 bue 1821 2451 130 075820 bue 2483 2958 10 1 CSF1PO blue 30009 3466 1042 0351358 green 98 0 145 6 14 15 D55818 green 1535 20449 11 D135317 green 2045 24901 11 0165539 green 2503 296 81 1112 0195433 0251338 green 30249 3680 1923 0195433 yellow 967 1437 1445 VINA yellow 1485 2110 1748 AMEL 0125381 yellow 2145 274 49 820 0651043 018551 yellow 27999 3655 15 9 FGA AMEL red 1073 1153 0651043 1186 2046 1218 Reference Samples red 20565 3594 2324 lt ii 4 7 Chapter 4 Analyzing Data To import panels and bin sets continued 8 View the markers and display the Bin view in the navigation pane a Select the Sinofiler_v1 folder to display its list of markers in the right pane b Double click the Sinofiler_v1 folder to display its list of markers below it c Select 0128391 to display the Bin view for the marker in the right pane IP Panel Manager File Edit Bins View FA AmpFLSTR Sinofiler Panels v1 AmpFLSTR Sinofier Bins v1 a b 5 _ 1 0851179 14 15 16 47 17 021511 075820 CSF1PO 0351358 055818 0135317 0165539 0251338 0195433 018551 AMEL 0651043 3 FGA Samples 9 Click Apply then OK to add the panel and bin set to the GeneMapper D database
85. like many other DNA polymerases can catalyze the addition of a single nucleotide predominately adenosine to the 3 ends of double stranded PCR products Clark 1988 Magnuson et al 1996 This nontemplate addition results in a PCR product that is one nucleotide longer than the actual target sequence The PCR product with the extra nucleotide 15 referred to as the A form AmpFSTR Sinofiler PCR Amplification Kit User Guide 5 23 Chapter 5 Experiments and Results The efficiency of A addition is related to the particular sequence of the DNA at the 3 end of the PCR product The Sinofiler kit includes two main design features that promote maximum addition The primer sequences have been optimized to encourage A addition The final extension step is 60 C for 60 min The final extension step gives the AmpliTaq Gold DNA polymerase additional time to complete A addition to all double stranded PCR products STR systems where each allele is represented by two peaks that are one nucleotide apart that have not been optimized for A addition may have split peaks 5 24 AmpF amp TR Sinofiler PCR Amplification Kit User Guide Extra Peaks in the Electropherogram 170 175 180 185 190 195 EM A No Extension Final Extension Figure 5 6 Omitting the final extension step results in split peaks due to incomplete A nucleotide addition Data are from an ABI PRISM 310
86. ling instructions Wear appropriate protective eyewear clothing and gloves Note Adjust the volumes accordingly for specific needs 2 Aliquot and autoclave the solutions 3 Store at room temperature AmpF amp TR Sinofiler PCR Amplification Kit User Guide 2 3 Chapter 2 PCR Amplification Quantifying DNA Importance of Quantitation Methods for Quantifying DNA By quantifying the DNA in a sample you determine if there is enough DNA for adequate amplification You can determine the smallest volume necessary to obtain 0 50 to 1 25 ng of DNA However the maximum allowable addition of DNA is 10 uL 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 Quantitation peak height and area for off scale peaks is not accurate For example an allele peak that 1s off scale can cause the corresponding stutter peak to appear higher in relative intensity thus increasing the calculated percent stutter Multicomponent analysis of off scale data is not accurate resulting in poor spectral separation pull up Incomplete A nucleotide addition When the total number of allele copies added to the PCR is extremely low allelic dropout can occur resulting in a partial profile
87. m 9700 AmpF amp TR Sinofiler PCR Amplification Kit User Guide 2 7 Chapter 2 PCR Amplification Performing PCR To run PCR Program the thermal cycling conditions IMPORTANT If using the Gold plated Silver or Silver 96 Well GeneAmp PCR System 9700 select the 9600 Emulation Mode Initial Cycle Final Final Incubation 28 cycles Extension Hold Step De Anneal Extend nature HOLD CYCLE HOLD HOLD 95 94 59 C 72 C 60 C 4 C 11 min 1 min 1 min 1 min 60 min Load the plate into the thermal cycler and close the heated cover ANTITE PHYSICAL INJURY HAZARD During instrument operation the temperature of the heated cover can be as high as 108 C and the temperature of the sample block can be as high as 100 C Keep hands away from the heated cover and sample block Start the run Store the amplified DNA If you are storing the DNA for Store at lt 2 weeks 2 8 gt 2 weeks 15 to 25 C IMPORTANT Protect the amplified products from light 2 8 AmpFSTR Sinofiler PCR Amplification Kit User Guide Amplification Using Bloodstained Cards Amplification Using Bloodstained FTA Cards FTA treated DNA collection cards can be useful for the collection storage and processing of biological samples A small punch of the bloodstained card can be placed directly into an amplific
88. m 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 Hu Y Liao M Zhou Y Zhang L Chen G D 2004 Polymorphisms of five short tandem repeat systems in Chinese Zang population in Kangba area Sichuan Da Xue Xue Bao Yi Xue Ban 35 21 24 Chinese Junge A and Madea B 1998 Validation studies and characterization of variant alleles at the short tandem repeat locus 0125391 Int Legal Med 112 67 69 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 AmpF STR Sinofiler PCR Amplification Kit User Guide Bibliography 3 Bibliography 4 Kimpton C Walton A and Gill P 1992 A further tetranucleotide repeat polymorphism in the vWF gene Hum Mol Genet 1 287 Klintschar M Ricci U al Hammadi N Reichenpfader B Ebner A Uzielli 1998 Genetic variation at the STR loci 0125391 and CSF1PO in four populations from Austria Italy Egypt and Yemen Forensic Sci Int 97 37 45 Kwok S and Higuchi R 1989 Avoiding false positives with PCR Nature 339 237 238 Lareu M V Pestoni C Sch renkamp M Rand S Brinkmann B and Carracedo A 1996 A highly variable STR
89. mine whether it is possible to resolve the genotypes of the major and minor component s at a single locus The ability to obtain and compare quantitative values for different allele peak heights on Applied Biosystems instruments provides an additional means to resolve mixed genotypes The quantitative value is much less subjective than comparing relative intensities of bands on a stained gel Ultimately the likelihood that any sample 1s a mixture must be determined by the analyst in the context of each particular case including the information provided from known reference sample s AmpF amp TR Sinofiler PCR Amplification Kit User Guide 5 39 Chapter 5 Experiments and Results 100 110 120 130 140 150 160 170 180 190 200 210 220 230 40 250 260 270 280 290 300 310 320 330 340 350 360 M 2000 0 400 410 120 430 440 10 40 410 140 10 200 210 20 230 240 250 260 270 280 290 300 310 320 330 340 350 360 e eae e je Kir E 4 300 15 1 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350 360 S TINTE a 100 110 120 130 140 450 160 170 180 130 200 210 220 230 240 250 260 270 280 230 300 310 320 330 340 350 360 200 Ti E x 3 1 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 2
90. minutes 8 Place the sample tray on the autosampler 9 Start the electrophoresis run 3 8 AmpFSTR Sinofiler PCR Amplification Kit User Guide Part Number 4384256 Rev 03 2012 Chapter 4 Analyzing Data AmpFSTR Sinofiler PCR Amplification Kit User Guide Analyzing Data This chapter covers Overview of GeneMapper ID Software 4 2 Setting Up GeneMapper ID Software for Analyzing AmpF STR Sinofiler Kit 4 3 Analyzing and Editing Sample Files with GeneMapper ID 4 17 AmpF amp TR Sinofiler PCR Amplification Kit User Guide 4 1 Chapter 4 Analyzing Data Overview of GeneMapper D Software GeneMapper JD Software is an automated genotyping software for forensic paternity and database data analysis and other genotyping needs After electrophoresis the data collection software stores information for each sample in a fsa file Using GeneMapper v3 2 1 and v3 3 software you can then analyze and interpret the data from the fsa files Instruments Refer to Instrument and Software Overview on page 1 7 for a list of compatible instruments Before You Start When using GeneMapper JD Software v3 2 1 and v3 3 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 Y
91. ngdu Zhonghua Yi Xue Yi Chuan Xue Za Zhi 16 77 80 Chinese Chen H Li X W Lian J H Song G Y Cheng X L Y Qi H Liu H Zhang Q X 2004 Genetic polymorphisms of six short tandem repeat loci in the Han population of Henan province in China Zhonghua Yi Xue Yi Chuan Xue Za Zhi 21 640 642 Chinese Chung D T Drabek J Opel K L Butler J M and McCord B R 2004 A study of the effects of degradation and template concentration on the amplification efficiency of the Miniplex primer sets J 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 Characterization of new miniSTR loci to aid analysis of degraded DNA J 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 DNA Advisory Board Federal Bureau of Investigation U S Department of Justice 1998 Quality assurance standards for forensic DNA testing laboratories Drabek J Chung D T Butler J M McCord 2004 Concordance study between Miniplex assays and a commercial STR typing kit J Forensic Sci 49 859 860 Edwards A Civitello A Hammond H and Caskey C 1991 DNA typing and genetic mapping with trimeric and tetramer
92. nherited as allele 18 single step mutation The maternal paternal source of this mutation could not be distinguished Additional Mutation Studies Additional studies Edwards et al 1991 Edwards et al 1992 Weber and Wong 1993 Hammond et al 1994 Brinkmann et al 1995 Chakraborty et al 1996 Chakraborty et al 1997 Brinkmann et al 1998 Momhinweg et al 1998 Szibor et al 1998 of direct mutation rate counts produced e Larger sample sizes for some of the AmpF STR Sinofiler PCR Amplification Kit loci Methods for modifications of these mutation rates to infer mutation rates indirectly for those loci where the rates are not large enough to be measured directly and or to account for those events undetectable as Mendelian errors 5 58 AmpF amp TR Sinofiler PCR Amplification Kit User Guide Probability of Identity Probability of Identity Table 5 6 shows the Probability of Identity PI values of the AmpF STR Sinofiler PCR Amplification Kit loci individually and combined Table 5 6 Probability of Identity values for the AmpF STR Sinofiler kit STR loci Locus pales Asian Caucasian Hispanic CSF1PO 0 074 0 119 0 127 0 142 D12S391 0 041 0 046 0 025 0 026 D13S317 0 145 0 069 0 077 0 059 D16S539 0 070 0 082 0 101 0 084 D18S51 0 030 0 039 0 031 0 029 D19S433 0 037 0 067 0 081 0 049 D21S11 0 040 0 07 0 052 0 049 02 1338 0 024
93. 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 AmpFSTR Sinofiler PCR Amplification Kit User Guide How to Obtain More Information How to Obtain More Information Related To obtain any of the following documents go to Documentation www appliedbiosystems com then click the links for Support gt Products amp Services Literature Part Document Number Applied Biosystems 3130 3100xl Genetic Analyzers Using Data Collection Software v3 0 4363787 User Bulletin Applied Biosystems 3130 3130xl Genetic Analyzers Getting Started Guide 4352715 Applied Biosystems 3130 3130 Genetic Analyzers Maintenance Troubleshooting and 4352716 Reference Guide Applied Biosystems 3130 3130xl Genetic Analyzers Quick Reference Card 4362825 Applied Biosystems 3130 3130xl Genetic Analyzers AB Navigator Software 4359472 Administrator Guide ABI 3100 3100 Avant Data Collection v2 0 User Guide 4347102 ABI 3100 3100 Avant Genetic Analyzers Using Data Collection Software v2 0 4350218 User Bulletin ABI 3100 Genetic Analyzer User Manual Data Collection v1 1 4315834 ABI
94. nofiler PCR Amplification Kit User Guide Weber J and Wong 1993 Mutation of human short tandem repeats Hum Mol Genet 2 1123 1128 Wiegand P and Kleiber M 2001 Less is more length reduction of STR amplicons using redesigned primers Int Legal Med 114 285 287 Wu S Z Zhang H Q Bi Y T 2004 Polymorphism of STR loci D128391 D18S865 in Wenzhou Han population Fa Yi Xue Za Zhi 20 85 7 Chinese Yu L Wu X Y Cai G Q Ou J H L M 2003 Sequence variation of 0125391 and 0118554 loci in Guangzhou han population Yi Chuan Xue Bao 30 781 4 Chinese AmpF amp TR Sinofiler PCR Amplification Kit User Guide Bibliography 7 Bibliography 8 AmpF amp TR Sinofiler PCR Amplification Kit User Guide Index Symbols A nucleotide addition defined 5 23 efficiency of 5 24 lack of causes 5 25 Numerics 310 allelic ladder requirements 3 2 3100 and 3130 series allelic ladder requirements 3 2 A A 2 9 accuracy and reproducibility 5 3 alleles low frequency 5 56 off ladder 5 4 peak height ratio table 5 37 allelic bin definitions 4 2 offsets 4 2 allelic ladder analysis method for 4 2 figure 1 5 number per run suggested 3 2 precision results table 5 6 requirements for accurate genotyping 3 2 sample type 4 2 volume per reaction 3 5 3 7 AmpFISTR_Panels_v3 folder 4 6 analysis method settings 4 11 analysis method for allelic ladders 4 2 analysis settings for project 4 17 Applied Bi
95. nofiler PCR Amplification Kit to label samples are 6 FAM NED and PET dyes The fifth dye LIZ is used to label the GeneScan 500 LIZ Size Standard Each of these fluorescent dyes emits its maximum fluorescence at a different wavelength During data collection on the Applied Biosystems and ABI PRISM instruments the fluorescence signals are separated by a 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 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 1 2 The goal of multicomponent analysis is to correct for spectral overlap Dyes 6 FAM VIC NED PET LIZ Normalized Emission 500 550 600 650 700 Wavelength nm Figure 1 2 Emission spectra of the five dyes used in the AmpF STR Sinofiler PCR Amplification Kit amp Sinofiler PCR Amplification Kit User Guide Materials and Equipment Materials and Equipment Kit Contents The AmpF STR Sinofiler kit contains sufficient quantities of the following reagents and the appropriate licenses to perform 200 25 uL Kit Storage and Stability amplifications
96. nown alleles have been published or reported to Applied Biosystems by other laboratories see the STRBase at http www cstl nist gov div831 strbase AmpF STR Table 5 5 shows AmpF STR Sinofiler kit allele frequencies in Sinofiler Kit four populations listed as percentages Allele WE Table 5 5 AmpF STR Sinofiler kit allele frequencies African i Allele American 1420 120 CSF1PO 6 7 5 56 0 68 0 951 8 6 98 0 451 0 371 0 711 9 4 44 4 32 2 21 2 37 10 24 44 22 95 29 04 23 70 11 23 65 25 91 29 60 30 81 12 27 30 37 73 29 96 36 97 13 6 83 6 82 7 90 3 55 14 0 79 0 68 0 92 0 95 15 p value 0 07 0 19 0 35 0 18 012 391 13 t 0 24 14 t AmpF amp TR Sinofiler PCR Amplification Kit User Guide 5 43 Chapter 5 Experiments and Results 5 44 Table 5 5 AmpF STR Sinofiler kit allele frequencies continued African American aeaa 1211 15 6 03 0 68 4 60 4 74 16 6 51 0 45 2 39 5 92 17 15 87 5 91 10 11 5 92 17 1 0 32 t t 17 3 0 48 2 21 1 42 18 25 71 24 55 15 62 17 54 18 3 0 95 0 454 2 02 1 90 19 16 98 18 18 10 66 18 25 19 1 0 63 t t t 19 3 0 16 t 0 55 2 84 20 10 95 17 5 12 32 13 51 20 3 t 0 23 0 37 0 24 21 6 83 10 68 13 05 10 66 22 3 33 9 32 13 42 9 24 23 2 06 5 9
97. ntrifuge the plate to ensure that the contents of each well are mixed and collected at the bottom Heat the reaction plate in a thermal cycler for 3 minutes at 95 ANITE PHYSICAL INJURY HAZARD During instrument operation the temperature of the heated cover can be as high as 108 C and the temperature of the sample block can be as high as 100 C Keep hands away from the heated cover and sample block Immediately place the plate on ice for 3 minutes Prepare the plate assembly on the autosampler Start the electrophoresis run AmpF amp TR Sinofiler PCR Amplification Kit User Guide 3 5 Chapter 3 Performing Electrophoresis Setting Up the 310 Instrument for Electrophoresis Reagents and Parts Electrophoresis Setup Software and Reference Documents Table 1 3 on page 1 11 lists the required materials not supplied with the AmpF STR Sinofiler PCR Amplification Kit 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 Sinofiler Allelic Ladder and GeneScan 500 LIZ Size Standard should also be protected from light Minimize freeze thaw cycles The following table lists data collection software and the run modules that you can use to analyze Sinofiler kit products For details on the analysis procedures refer to the documents listed in the table Operating System Data
98. oard 1998 The DAB standards describe the quality assurance requirements that a laboratory should follow to ensure the quality and integrity of the data and competency of the laboratory Additional validation was performed according to the revised guidelines from the Scientific Working Group on DNA Analysis Methods SWGDAM July 10 2003 Based on these guidelines Applied Biosystems conducted experiments that comply with guidelines 1 0 and 2 0 and its associated subsections This DNA methodology is not novel Moretti et al 2001 Frank et al 2001 Wallin et al 2002 and Holt et al 2000 This chapter discusses many of the experiments performed by Applied Biosystems and provides examples of results obtained Applied Biosystems chose conditions that produced maximum PCR product yield and that met reproducible performance standards It is our opinion that while these experiments are not exhaustive they are appropriate for a manufacturer Each laboratory using the AmpF STR Sinofiler PCR Amplification Kit should perform internal validation studies amp Sinofiler PCR Amplification Kit User Guide Accuracy Precision and Reproducibility Accuracy Precision and Reproducibility SWGDAM Developmental validation is the demonstration of the accuracy Guideline 1 2 1 precision and reproducibility of a procedure by the manufacturer technical organization academic institution government laboratory or other part
99. ograms from a species specificity study including positive and non template controls NTC 5 30 AmpF amp TR Sinofiler PCR Amplification Kit User Guide Species Specificity Figure 5 8 on page 5 30 shows amplification for control DNA 9947A 1 ng panel 1 chimpanzee 1 ng panel 2 pig 10 ng panel 3 cat 10 ng panel 4 microbial DNA pool equivalent to 10 copies of Candida albicans Neisseria gonorrhoeae E coli 0157 H7 Bacillus subtilis and Lactobacillus rhamnosus panel 5 and the negative control panel 6 The extracted DNA samples were amplified with the Sinofiler kit and analyzed using the Applied Biosystems 3130x Genetic Analyzer Primates gorilla chimpanzee orangutan and macaque 1 ng each Non primates mouse dog pig cat horse hamster rat chicken and cow 10 ng each Microorganisms Candida albicans Staphylococcus aureus Escherichia coli Neisseria gonorrhoeae Bacillus subtilis and Lactobacillus rhamnosus equivalent to 10 copies All the primate DNA samples amplified producing fragments within the 100 to 400 base pair region Lazaruk et al 2001 Wallin et al 1998 The microorganisms chicken cat hampster rat and mouse did not yield detectable product Horse cow dog and pig produced a 104 bp fragment near the amelogenin locus in PET dye AmpF amp TR Sinofiler PCR Amplification Kit User Guide 5 31 Chapter 5 Experiments and Results Sensitivity SWGDAM
100. om more than one individual are an integral component of forensic casework Therefore it is essential to ensure that the DNA typing system must be able to detect DNA mixtures Mixed samples can be distinguished from single source samples in a variety of ways The presence of greater than two alleles at a locus The presence of a peak at a stutter position that is significantly greater in percentage than what is typically observed in a single source sample Significantly imbalanced alleles for a heterozygous genotype AmpF amp TR Sinofiler PCR Amplification Kit User Guide 5 37 Chapter 5 Experiments and Results The peak height ratio is defined as the height of the lower peak in RFU divided by the height of the higher peak in RFU expressed as a percentage Mean median minimum and maximum peak height ratios observed for alleles the AmpF STR Sinofiler PCR Amplification Kit loci in unmixed population database samples are shown inTable 5 3 Table 5 3 Peak height ratios for 1 ng of input DNA Number of Allele n Mean Median Minimum Maximum CSF1PO 581 88 8 90 4 55 8 99 9 0125391 688 87 9 89 2 57 4 100 0135317 590 88 4 89 3 59 5 99 9 0165539 621 88 6 89 8 52 4 100 018551 703 87 6 88 3 57 5 100 019 433 637 89 8 90 6 62 6 100 021 11 689 89 4 90 8 62 3 100 02 1338 702 87 2 88 8 45 1 99 9 03 1358 584 90 5 92 0 57 5 99 9 05 818 575 89 9 91 3 62 0 100 06 1043 674 90 0 91
101. or 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 CHEMICAL HAZARD Hi Di Formamide Exposure causes eye skin and respiratory tract irritation It is a possible developmental and birth defect hazard Read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves 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 25 uL of the formamide size standard mixture 1 5 uL of PCR product or Allelic Ladder AmpF amp TR Sinofiler PCR Amplification Kit User Guide 3 7 Chapter 3 Performing Electrophoresis To prepare samples for electrophoresis continued 5 Seal the tubes with appropriate septa then briefly centrifuge the tubes to ensure that the contents of each tube are mixed and collected at the bottom 6 Heat the tubes in a thermal cycler for 3 minutes at 95 C ANITE PHYSICAL INJURY HAZARD During instrument operation the temperature of the heated cover can be as high as 108 C and the temperature of the sample block can be as high as 100 C Keep hands away from the heated cover and sample block 7 Immediately place the tubes on ice for 3
102. osystems contacting xii customer feedback on documentation xii AmpFSTR Sinofiler PCR Amplification Kit User Guide Information Development department xii Technical Support xii artifacts in data 5 26 B baseline noise examples 5 27 bin sets importing 4 6 viewing 4 8 Bin view displaying for a marker 4 8 biohazardous waste handling 1 Bloodstained Cards amplification 2 9 bold text when to use v C CAUTION description vi CEPH 5 28 characterization of loci validation 5 28 chemicalsafety chemical waste safety 1 concordance studies 5 57 contents of kit 1 9 control DNA about 1 10 control DNA about 1 10 conventions bold text for describing menu commands IMPORTANTS v italic text v Notes v user attention words v customer feedback on Applied Biosystems documents xii Index 1 DANGER description vi data collection software 1 7 data accuracy precision and reproducibility of 5 3 data artifacts 5 26 data for different populations 5 42 developmental validation 5 2 DNA degraded 5 34 effect of quantity on result 5 32 effect of quantity figure 5 33 methods for quantifying 2 4 mixture studies 5 37 mixture studies figure 5 39 negative control sample preparation 2 7 positive control sample preparation 2 7 quantitation importance of 5 32 sensitivity 5 32 stability 5 34 your sample preparation 2 7 DNA mixtures amplification figure 5 40 limit of detection 5 40 documentation
103. ote Ifa sample allele peak is called as an off ladder allele the sample result needs to be verified according to the laboratory s protocol Setting Up GeneMapper D Software for Analyzing AmpF STR Sinofiler Kit Data Workflow Before you can analyze sample fsa files using GeneMapper Software v3 2 1 or v3 3 for the first time you need to Import panels and bins into the Panel Manager as explained in Importing Panels and Bins 3 2 1 only on page 4 4 This section does not apply to GeneMapper ID Software v3 3 which automatically installs panels and bin sets during installation Import an analysis method as explained in Importing an HID Analysis Method v3 2 1 and v3 3 on page 4 9 Import a size standard as explained in Importing an HID Size Standard v3 2 1 and v3 3 on page 4 16 Define custom views of analysis tables v 3 2 1 and v3 3 Refer to Chapter 1 of the GeneMapper ID Software Versions 3 1 and 3 2 Human Identification Analysis Tutorial PN 4335523 for more information Define custom views of plots v 3 2 1 and v3 3 Refer to Chapter 1 of the GeneMapper ID Software Versions 3 1 and 3 2 Human Identification Analysis Tutorial PN 4335523 for more information If necessary convert any GeneScan software sample files generated on the Macintosh platform to the fsa format using the Mac to Win AppleScript software provided with GeneMapper software Conversion is described in the GeneMapper
104. otechniques 21 700 709 AmpF4STR Sinofiler PCR Amplification Kit User Guide 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 P D Budowle 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 Momhinweg E Luckenbach C Fimmers R and Ritter H 1998 0381358 sequence analysis and gene frequency a German population Forensic Sci Int 95 173 178 Moretti T Baumstark A Defenbaugh D Keys K Smerick J and Budowle B 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 Revised Validation Guidelines Scientific Working Group on DNA Analysis Methods SWGDAM Forensic Science Communications July 2004 Volume 6 3 Available at www fbi go
105. our laboratory can use multiple ladder samples in an analysis provided individual laboratories conduct the appropriate validation studies For multiple ladder samples the GeneMapper JD 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 e Allelic bin definitions are stored in the AmpF STR Sinofiler panels in the Panel Manager Lanes or 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 4 2 AmpFSTR Sinofiler PCR Amplification Kit User Guide Setting Up GeneMapper ID Software for Analyzing amp Sinofiler Kit Data e Alleles that are not in the AmpF STR Allelic Ladders do exist Off ladder 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 N
106. pF amp TR Sinofiler PCR Amplification Kit User Guide Extra Peaks in the Electropherogram 0851179 75820 5 1 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 0351358 10255818 10135317 10165539 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 M pt tt A sos 7 FGA 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 Figure 5 7 Examples of baseline noise and reproducible artifacts in data produced on the Applied Biosystems 3130x Genetic Analyzer Note that a high degree of magnification y axis is used to illustrate these artifacts AmpF amp TR Sinofiler PCR Amplification Kit User Guide 5 27 Chapter 5 Experiments and Results Characterization of Loci SWGDAM Guideline 2 1 Nature of the Polymorphisms 5 28 Inheritance The basic characteristics of a genetic marker must be determined and documented SWGDAM July 2003 This section describes basic characteristics of the 15 loci and the sex determining marker amelogenin that are amplified with the AmpF STR Sinofiler PCR Amplification Kit These loci have been extensively characterized by other laboratories The primers for the amelogenin locus flank a 6 nucleotide deletion within intron 1 of the X homologue Amplific
107. particular applications reagents and substrates used in your laboratory Ensure the health and safety of all personnel in your laboratory Ensure that the instrument waste is 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 TR Sinofiler PCR Amplification Kit User Guide ix Preface Biological Hazard Safety ANCE BIOHAZARD Biological samples such as tissues body fluids 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 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 US Department of Health and Human Services guidelines published in Biosafety in Microbiological and Biomedical Laboratories stock no 017 040 00547 4 bmbl od
108. resis on the 310 Instrument 3 7 Analyzing Data Overview of GeneMapper ID 4 2 Setting Up GeneMapper D Software for Analyzing AmpF STR Sinofiler Kit Data 4 3 Analyzing and Editing Sample Files with GeneMapper ID 4 17 Experiments and Results OVENI WA i adu oie sats Aad dod mete Mag wee eo 5 2 Accuracy Precision and 5 3 Extra Peaks in the Electropherogram 5 18 Characterization of Loci 5 28 Species Specificity 5 30 duos APA PLAT epRPewEPIOerie idu 5 32 beo eT eme UENIRE 5 34 Mixtur Studies awe nae ch tea pepe xi e EM C ER 5 37 Population Data 5 42 Mutation 5 58 Probability of Identity 5 59 Probability of Paternity Exclusion 5 61 Troubleshooting Troubleshooting A 2 AmpF amp TR Sinofiler PCR Amplification Kit User Guide Preface How to Use This Guide Purpose of This Applied Biosystems AmpF STR Sinofiler PCR Amplification Guide User Guide provides information about the Applied Biosys
109. rmware 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 1s then analyzed by the analysis software Operatin Data Instrument Collection Analysis Software y Software 3130 3130x Windows 3 0 GeneMapper D v3 2 1 and later 3100 3100 Windows NT 1 1 3100 GeneMapper D v3 2 1 Avant 1 0 3100 and later Avant Windows 2000 2 0 GeneMapper D v3 2 1 and later 310 Windows XP 3 1 GeneMapper D v3 2 1 and later Windows NT 3 0 GeneMapper D v3 2 1 and and later Windows 2000 t Applied Biosystems conducted validation studies for the Sinofiler kit using these configurations Applied Biosystems fluorescent multi color dye technology allows the analysis of multiple loci including loci that have alleles with overlapping size ranges Alleles for overlapping loci are distinguished by labeling locus specific primers with different colored dyes AmpFSTR Sinofiler PCR Amplification Kit User Guide 1 7 Chapter1 Overview How Multicomponent Analysis Works Multicomponent analysis is the process that separates the five different fluorescent dye colors into distinct spectral components The four dyes used in the AmpF STR Si
110. roject v3 2 1 and v3 3 1 In the Project window select File 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 Parameter Advanced Analysis Method Sample Type Select the sample type Analysis Method v3 2 1 Sinofiler HID v1 Analysis Method v3 3 Sinofiler HID v1 33 Panel v3 2 1 AmpFLSTR Sinofiler Panels v1 Panel v3 3 AmpFLSTR Sinofiler Panels v1 33 Size Standard CE G5 Sinofiler GS500 Matrix ek a matrix for 310 instruments only For more information about how the Size Caller works refer to the ABI PRISM GeneScan Analysis Software for the Windows NT9 Operating System Overview of the Analysis Parameters and Size Caller User Bulletin PN 4335617 The following fragments are defined for the CE G5 Sinofiler GS500 size standard provided with the AmpF STR kits 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 PN 4338775 Appendix D Neither the 250 nt nor the 340 nt peak are included in the size standard definition These peaks can be used as an indicator of precision within a run AmpF amp TR Sinofiler PCR Amplification Kit User Guide 4 17 Chapter 4 Analyzing Data 4 18 To analyze a project continued
111. sed to generate the population data provided in this section Samples were collected from individuals throughout the United States with no geographical preference Analysis across the three databases of 2 034 total chromosomes per locus revealed the following number of different alleles 8 CSFIPO alleles 14 0251338 alleles 12 D3S1358 alleles 9 055818 alleles 25 0651043 alleles 12 075820 alleles 11 0851179 alleles 21 0125391 alleles 10 D13S317 alleles 8 0165539 alleles 23 018551 alleles 19 D19S433 alleles 24 D21S11 alleles 28 FGA alleles and 12 vWA alleles Conformity of the observed genotype frequencies with Hardy Weinberg expectations HWE was examined in each sample population by the exact test using the HW QuickCheck software Bonferroni 1936 Guo and Thompson 1992 Kalinowski 2006 A p value 20 05 was obtained for all STRs except 0651043 in African American 0351358 in Asian 0195433 in Caucasian and D128391 in Hispanic samples Considering the Bonferroni procedure and the fact that 15 tests for HWE were simultaneously performed on AmpF amp TR Sinofiler PCR Amplification Kit User Guide Population Data the same population sample the significance threshold is adjusted from 0 05 to 0 05 15 0 0033 which is clearly below the p values observed in these loci Hence the departures from HWE were not significant In addition to the alleles that were observed and recorded in the Applied Biosystems databases other k
112. tate inter locus spacing The combination of a five dye fluorescent system and the inclusion of non nucleotide linkers allows for simultaneous amplification and efficient separation of the 15 STR loci and amelogenin during automated DNA fragment analysis AmpFSTR Sinofiler PCR Amplification Kit User Guide Loci Amplified by the Kit Product Overview Table 1 1 shows the loci amplified their chromosomal locations and the corresponding fluorescent marker dyes The AmpF STR Sinofiler Allelic Ladder is used to genotype the analyzed samples The alleles contained in the allelic ladder and the genotype of the Control DNA 99474 are also listed in the table Table 1 1 AmpF STR Sinofiler PCR Amplification Kit loci and alleles Locus Chromosome Alleles Included in Sinofiler Dve Label Control Designation Location Allelic Ladder y DNA 9947A D8S1179 8 8 9 10 11 12 13 14 15 16 6 FAM 13 17 18 19 021 11 21411 2 421 24 24 2 25 26 27 28 28 2 29 30 29 2 30 30 2 31 31 2 32 32 2 33 33 2 34 34 2 35 35 2 36 37 38 D7S820 7411 21 22 6 7 8 9 10 11 12 13 14 15 10 11 CSF1PO 5433 3 34 6 7 8 9 10 11 12 13 14 15 10 12 0351358 3p 12 13 14 15 16 17 18 19 14 15 055818 5421 31 7 8 9 10 11 12 13 14 15 16 11 0135317 13422 31 8 9 10 11 12 13 14 15 11 0165539 16q24 qter 5 8 9 10 11 12 1
113. tems instruments chemistries and software associated with the AmpF STR Sinofiler PCR Amplification Kit Pull Out Chapters This guide is designed to allow users to pull out chapters 2 3 and 4 The pull out chapters have title and back pages which indicate the chapter number and title Text Conventions This guide uses the following conventions Bold text indicates user action For example Type 0 then press Enter for each of the remaining fields e talic text indicates new or important words and is also used for emphasis For example Before analyzing always prepare fresh matrix A right arrow symbol gt separates successive commands you select from a drop down or shortcut menu For example Select File Open Spot Set Right click the sample row then select View Filter gt View All Runs User Attention Two user attention words appear in Applied Biosystems user Words documentation Each word implies a particular level of observation or action as described below Note Provides information that may be of interest or help but is not critical to the use of the product IMPORTANT Provides information that is necessary for proper instrument operation accurate chemistry kit use or safe use of a chemical AmpF amp TR Sinofiler PCR Amplification Kit User Guide v Preface Safety Safety Alert Words Chemical Hazard vi Warning Examples of the user attention words appear below Note
114. tion of a 7 locus multiplex STR test for use in forensic casework II 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 Gilliam T C 1993 A microsatellite genetic linkage map of human chromosome 18 Genomics 15 48 56 Szibor R Lautsch S Plate I Bender K and Krause D 1998 Population genetic data of the STR HumD3S1358 in two regions of Germany Intl Legal Med 111 160 161 Su X Li D Lifu S L Yun PW Yun S 2004 Population studies on two native Mongolian population groups in China using STR loci Forensic Sci Int 141 197 199 Waiyawuth W Zhang L Rittner C Schneider PM 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 PS 1998 SWGDAM validation of the AmpFISTR 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 Forensic Sci 47 52 65 Walsh P S Fildes N J Reynolds 1996 Sequence analysis and characterization of stutter products at the tetranucleotide repeat locus Nucleic Acids Res 24 2807 2812 AmpF amp TR Si
115. ts of hematin 5 35 Chapter 5 Experiments and Results 100 110 120 130 140 150 160 170 180 130 200 210 220 230 240 250 260 270 280 290 300 310 320 330 350 2000 40 uM 4600 Hematin 2000 50 uM 1600 Hematin 1200 800 400 0 lt 20001 60 uM Hematin 1200 s00 2400 2000 70 uM 4600 Hematin 4200 800 400 0 Figure 5 11 Amplification with the AmpF STR Sinofiler kit in the presence of varying concentrations of hematin analyzed on the Applied Biosytstems 3130x Genetic Analyzer 5 36 AmpF amp TR Sinofiler PCR Amplification Kit User Guide Mixture Studies Mixture Studies SWGDAM Guideline 2 8 Mixture Studies The ability to obtain reliable results from mixed source samples should be determined SWGDAM July 2003 Evidence samples may contain DNA from more than one individual The possibility of multiple contributors should be considered when interpreting results Applied Biosystems recommends that individual laboratories assign a minimum peak height threshold based on validation experiments performed in each laboratory This practice avoids typing when stochastic effects are likely to interfere with accurate interpretation of mixtures Evidence samples that contain body fluids and or tissues originating fr
116. v hq lab fsc current standards 2004 03 standards02 htm 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 J Hum Genet 75 1143 1148 Lareu M V Pestoni M C Barros F Salas A Carracedo A 1996 Sequence variation of a hypervariable short tandem repeat at the 0129391 locus Gene 182 151 153 Sensabaugh G F 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 AmpF amp TR Sinofiler PCR Amplification Kit User Guide Bibliography 5 Bibliography 6 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 J Legal Med 109 186 194 Sparkes R Kimpton Gilbard 5 Carne P Andersen J Oldroyd N Thomas D Urquhart A and Gill P 19965 The valida
117. vided by the chemical manufacturer before you store handle or work with any chemicals or hazardous materials See About MSDSs on page vii 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 MSDS 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 MSDS Check regularly for chemical leaks or spills If a leak or spill occurs follow the manufacturer s cleanup procedures as recommended in the MSDS Comply with all local state provincial or national laws and regulations related to chemical storage handling and disposal About MSDSs Chemical manufacturers supply current Material Safety Data Sheets MSDSs with shipments of hazardous chemicals to new customers They also provide MSDSs with the first shipment of a hazardous chemical to a customer after an MSDS has been updated MSDSs provide the safety information you need to store handle transport and dispose of the chemicals safely Each time you receive a new MSDS packaged with a hazardous chemical be sure to replace the appropriate MSDS in your files Obtaining The MSDS for any chemical supplied by Applied Biosystems is MSDSs available to you free 24 hours a day To obtain MSDSs
118. xl Genetic Analyzers Maintenance Troubleshooting and Reference Guide PN 4352716 AmpF amp TR Sinofiler PCR Amplification Kit User Guide 1 11 Chapter 1 Overview Table 1 3 User supplied materials continued Item Source 3100 3100 Avant Analyzer materials 96 Well Plate Septa 4315933 Reservoir Septa 4315932 3130 3100 Genetic Analyzer Capillary Array 36 cm 4315931 3130 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 uL Glass Syringe array fill syringe 4304470 5 0 mL Glass Syringe polymer reserve syringe 628 3731 For a complete list of parts and accessories for the 3100 3100 Avant instrument refer to Appendix B of the ABI PRISM 3100 Genetic Analyzer and 3100 Avant Genetic Analyzer User Reference Guide PN 4335393 310 Analyzer materials 310 Genetic Analyzer Capillary 47 cm 402839 0 5 mL Sample Tray 5572 96 Well Tray Adapter for 9700 thermal cycler trays 4305051 GeneScan 500 LIZ Size Standard 4322682 Running Buffer 10x 402824 Genetic Analyzer Septa Retainer Clips for 96 Tube Sample Tray 402866 Genetic Analysis Sample Tub
119. y SWGDAM July 2003 SWGDAM extent to which a given set of measurements of the same sample Guideline 2 9 agree with their mean and the extent to which these measurements match the actual values being measured should be determined SWGDAM July 2003 Accuracy Laser induced fluorescence detection of length polymorphism at short tandem repeat loci is not a novel methodology Holt et al 2000 and Wallin et al 2002 However accuracy and reproducibility of AmpF STR Sinofiler PCR Amplification Kit profiles have been determined from various sample types Figures 5 1 illustrates the size differences that are typically observed between sample alleles and allelic ladder alleles on the Applied Biosystems 3130x Genetic Analyzer with POP 4 polymer The x axis in Figure 5 1 on page 5 4 represents the nominal nucleotide sizes for the AmpF STR Sinofiler Allelic Ladder The dashed lines parallel to the x axis represent the 0 25 nt windows The y axis represents the deviation of each sample allele size from the corresponding allelic ladder allele size All sample alleles are within 0 5 nt from a corresponding allele in the allelic ladder AmpF amp TR Sinofiler PCR Amplification Kit User Guide 5 3 Chapter 5 Experiments and Results CSF1PO 02 1338 D381358 058818 0651043 075820 0851179 0125391 0135317 0165539 018551 0195433 021511 WA AMEL o A n 4 9 o o Si
120. ze Difference nt 120 140 160 180 200 220 240 260 280 300 320 340 360 Allele Size nt Figure 5 1 Size deviation of 54 samples analyzed on the Applied Biosystems 3130x Genetic Analyzer Precision Sizing precision allows for determining accurate and reliable Size Windows genotypes Sizing precision was measured on the Applied Biosystems 3130x Genetic Analyzer The recommended method for genotyping is to employ a 0 5 nt window around the size obtained for each allele in the AmpF STR Sinofiler Allelic Ladder A 0 5 nt window allows for the detection and correct assignment of alleles Any sample allele that sizes outside the specified window could be An off ladder allele that is an allele of a size that is not represented in the AmpF STR Sinofiler Allelic Ladder or An allele that corresponds to an allelic ladder allele but whose size is just outside a window because of measurement error 5 4 AmpFSTR Sinofiler PCR Amplification Kit User Guide Accuracy Precision and Reproducibility The measurement error inherent in any sizing method can be defined by the degree of precision in sizing an allele multiple times Precision is measured by calculating the standard deviation in the size values obtained for an allele that is run in several injections on a capillary instrument or in several lanes of one gel Table 5 1 on page 5 6 shows typical precision results obtained from five runs

AmpFlSTR Sinofiler PCR Amplification Kit User Guide (PN 4384256C)

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