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AmpFlSTR® MiniFiler™ PCR Amplification Kit User Guide (PN

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1. 19 DNA guat Cat OD e 5 2 e et BM M gt e pa Ph ane IDI V Z 19 Importance of quantification 19 Methods of quantifying 20 Prepare the amplification kit 5 21 decens aida te dya a A EET MEE M 22 Amplification using bloodstained FTA cards 22 CHAPTERS Perform Electrophoresis 25 Allelic ladder requirements 26 Section 3 1 3100 3100 Avant and 3130 3130xl instruments 27 Set up the 3100 3100 Avant and 3130 3130xl instruments for electrophoresis 27 Reagents erp Up COLIMA DAN DANI excede bed 27 Electrophoresis software setup and reference documents 27 Prepare samples for electrophoresis on the 3100 3100 Avant or 3130 3130xl instruments 28 AmpFtSTR MiniFiler PCR Amplification Kit User Guide 3 Contents Section 3 2 3500 3500xL instruments 29 Set up the 3500 3500xL instruments for 29 Reagents and patts AER ets dec te to e te
2. 38 Allele tab setting Surni ee SEK A 39 Peak Detector tab settings 40 Peak Quality tab 05 41 Quality Flags tabssettings icio am Rb x Rp REESE Robe e ead 42 Create a size standard 43 Analyze edit sample files with GeneMapper ID Software 44 Examine and edit 45 For more information 45 Section 4 2 GeneMapper D X Software 46 Overview of GeneMapper D X Software 46 DD M anti eye EE 46 Before yourstart te cest ele oe eb beds s teen 46 Set up GeneMapper D X Software for data analysis 47 Panel bin and stutter file version 47 Before using the software for the 47 Check panel bin and stutter file version 47 Import panels bi
3. 105 Degraded DNA 5 kk kk mE kk kl VAT epu 105 Intracolorbalane i car n aka ERR hs RE eh 105 Mean referenced peak 106 J pp LL M MI LI gg 107 Inhibition Study ca HHHH HHHH HH RR HH HHHH tet vie te 108 Mean peak height minimum mean peak height and intracolor 108 Allelicdropoub Gatien Yo Sales He 112 CONCIUSION sc S NIRE 112 APPENDIX A Troubleshooting 113 APPENDIX 3rd Order Least Squares Sizing Method 115 When io Use ree IA TEX YN 115 About the Local Southern algorithm WWW kk kk kk kk kk kk kk kK KK KK KK kK KK KK KK KK KK KK KK kk kk 115 Comparing genotyping 116 AmpFtSTR MiniFiler PCR Amplification Kit User Guide Contents APPENDIX Ordering Information 117 Equipment and materials not 117 APPENDIX D PCR Work 121 Work area setup and lab 121 PCR setup work ar
4. 40004 1 30 20004 4 200 70 80 30 100 110 120 130 140 150 160 170 180 130 200 210 220 230 240 250 260 270 280 230 40004 0 M 20 0 70 80 30 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 rc ti ti tts ZEL ZEDE ERKA ZE axa Zana 40004 0 1 NE 10 0 70 80 30 100 110 120 130 140 150 160 170 180 130 200 210 220 230 240 250 260 270 280 290 70 80 90 400 440 420 430 440 450 460 180 490 200 210 220 200 250 260 200 200 280 Ir A 40001 20001 10 0 t iri 70 80 90 400 410 420 430 440 450 460 470 180 490 210 220 230 200 250 900 270 200 280 40001 1 _9no zel 20 i i vy i T 1p 20 220 230 240 250 260 270 280 290 Thermal cycler Thermal cycling parameters were established for amplification of the MiniFiler Kit parameters Thermal cycling times and temperatures of GeneAmp PCR systems were verified Varying annealing and denaturation temperature win
5. 2 94 Mutation Tate remite uet ke hy Dl Wa KU RR arka a Qebe gua Welal a a kana MI UR AUTO 95 Estimating germ line mutations 95 Additional mutation studies 95 Probability of identity px RR pase sax n d pd ERR ER 95 Probability of paternity exclusion 96 Section 5 2 Performance Validation After Buffer and Enzyme Component Replacement a DE ent hg cut pud MM 98 LAY DO eSI REL LIEU ede 98 ExpeLiimiente s s iub nets eee Rb e ote ad Re SES otras 98 Reproducibility study m me REBEL A ka SIRAC neg Ale 99 Intracolor balance 22 veo luc er Re bau Rr bee Reed YR ed 99 St tter Dercentades ott NS Susa Da u te a Siy kek b 99 Pour teen Pena terror treet rt Pun tre PAD EUER 100 Sensitivity Study ioc ved op Peele dea we OME ein Pee 101 Mean referenced peak 101 DNA concentration and peak 102 Allelic dropout cien RAQ E cob 103 Genotype concordance
6. E a D 9 8 5 E S gi 1 v 1 s e lt 7 oad D H E Man 5 IE 4 2 09 1 4 i i Hl 3 i 3 2 3 i 0 5 6 7 8 9 10 11 12 13 14 15 16 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 D16S539 D18S51 Figure 11 Stutter percentages for the CSF1PO and FGA loci 18 17 16 15 14 s 13 JR 12 z 11 5 L4 4 1 9 ar 2 5 8 Je d i Hl I JE 7 HE i i f ti 6 T o g 1 5 2i A t 1 1 Hun 4 Li offs oft s 1 3 f 2 te l 1 0 5 6 7 8 9 10111213141516 16 17 18 19 20 2122 23 24 25 26 27 28 29 30 3132 33 3435 42 43 44 45 46 47 48 49 50 51 52 CSF1PO FGA AmpFtSTR9 MiniFiler PCR Amplification Kit User Guide 77 78 Chapter 5 Experiments and Results Extra peaks in the electropherogram Table 4 Marker specific stutter filter percentages for MiniFiler Kit loci Locust Stutter D13S317 14 D7S820 11 D2S1338 18 D21S11 16 D16S539 15 D18S51 18 CSF1PO 14 FGA 15 t These percentages are used as stutter filters in GeneMapper ID v3 2 1 AmpFLSTR MiniFiler Panels v1 txt and D X AmpFLSTR Stutter v2X IMPORTANT The values shown are the values we determined during developmental validation studies We recom
7. 9 S af Panel Manager Panel Name Comment E AmpFLSTR_NGMSElect_v2X Ofiler v1 1X null 4 SGM_Plus_v1 1X null AmpFLSTR _Panels_v1x IM SElect v2 1X Il AmpFLSTR Panels v2X SElect v2 1 m Identifiler Plus v1 1X null NGM v3 1X null Identifier Direct v1 1X null b Select File Import Bin Set to open the Import Bin Set dialog box c Navigate to then open the AmpFLSTR Analysis Files GMIDX folder AmpFtSTR9 MiniFiler PCR Amplification Kit User Guide 49 Chapter 4 GeneMapper ID X Software Set up GeneMapper ID X Software for data analysis d Select AmpFLSTR_Bins_v2X txt then click Import Note Importing this file associates the bin set with the panels in the AmpFLSTR_Panels_v2X folder Import Bin Set Look in AmpFLSTR Analysis Files GMIDX AmpFLSTR Panels v2x My Recent Bl AmpFLSTR_Stutter_v2 Documents ReadMe_AmpFLSTR_v2x My Documents 48 Filename AmpFLSTR Bins v2X bxt My Computer Files of type all Files 7 View the imported panels in the navigation pane a Double click the AmpFLSTR Panels v2X folder b Double click the MiniFiler v1 1X folder to display the panel information in the right pane Panel Manager File Edit Bins View Help N EN AmpFLSTR_Bins_ v i W Be S
8. Chapter 5 Experiments and Results Developmental validation PCR components We examined the concentration of each component of the MiniFiler Kit and established that the concentration of each component was within the range where data indicated that the amplification met the required performance criteria for specificity sensitivity and reproducibility For example various magnesium chloride concentrations were tested on the Applied Biosystems 3130 Genetic Analyzer The amplification of 0 50 ng of the control DNA 007 is shown in Figure 4 on page 66 We observed that the performance of the multiplex is most robust within a 20 window of magnesium chloride For example blood and buccal samples on treated paper substrates or swab sample lysates were amplified in the presence of varying concentrations of magnesium chloride and the results were analyzed on an Applied Biosystems 3130x or 3500xL Genetic Analyzer Results are shown in Figure 4 The performance of the multiplex is robust within 20 of the optimal magnesium chloride concentration Figure 4 A 0 50 ng sample of Contol DNA 007 amplified with the MiniFiler Kit in the presence of varying concentrations of magnesium chloride and analyzed on an Applied Biosystems 3130xl Genetic Analyzer y axis 5000 RFU 70 80 30 100 110 120 130 140 150 160 170 180 430 210 220 230 2 250 260 270 280 290 _ _ 4
9. 20004 Control Mark Sample for Deletion 75 95 115 135 155 175 195 215 235 255 3000 2000 Control B 10004 Mark Sample for Deletion 75 95 115 135 155 175 195 215 235 255 3000 2000 1000 U e ED 3 3 hy 2 e 0 5 ge 2 00 e ra 2 oH m N lt 3 9 2 9 Em 5 e D 3 D 5 Mark Sample for Deletion 75 95 415 135 155 175 195 215 235 255 3000 2000 Test B 1000 0 Mark Sample for Deletion 75 95 415 135 155 175 195 245 235 255 3000 AmpFtSTR MiniFiler PCR Amplification Kit User Guide 111 Chapter 5 Performance Validation After Buffer and Enzyme Component Replacement Conclusions Figure 37 Inhibition study representative electropherograms using Control DNA 007 inhibited with 50 ng pL Humic Acid Y scale 4000 RFU 75 3000 95 115 135 155 175 195 215 235 255 2000 1000 75 3000 Control Mark Sample for Deletion 95 115 135 155 175 195 215 235 255 E Control B 1000 75 30004 E Mark Sample for Deletion 95 115 135 155 175 195 215 235 255 75 3000 ie Test A 1000 Mark Sample for Deletion 95
10. Redilute DNA using TE Buffer with 0 1 mM EDTA AmpFtSTR MiniFiler PCR Amplification Kit User Guide 113 Appendix A Troubleshooting Observation Possible causes Recommended actions More than two alleles 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 0 75 ng Mixed sample Amplification of stutter product 1 repeat unit position See Stutter products on page 75 Incomplete 3 A base addition n 1 nt position See Experiments and Results on page 63 Be sure to include the final extension step of 60 C for 5 minutes in the PCR Signal exceeds dynamic range of instrument off scale data Ensure cycle number is optimized according to instructions on page 19 Repeat PCR amplification using fewer PCR cycles or use your laboratory s SOP to analyze off scale data Poor spectral separation bad matrix Follow the steps for creating a spectral file Confirm that Filter Set G5 modules are installed and used for analysis Some but not all loci visible on electropherogram of DNA Test Samples Test sample DNA is severely degraded If possible evaluate the quality of DNA sample by running an agarose gel If DNA is degraded re amplify with an increased amount of DNA Test sample co
11. GeneMapper D Software for data analysis File names Before using the software for the first time Import panels and bins 34 The file names shown in this section may differ from the file names you see when you download or import files If you need help determining the correct files to use contact your local Life Technologies Human Identification representative or go to www lifetechnologies com support gt Software Patches amp Updates GeneMapper ID Software To analyze sample files fsa using GeneMapper ID Software v3 2 1 for the first time Import panels and bins into the Panel Manager as explained in Import panels and bins on page 34 Create an analysis method as explained in Create an analysis method on page 38 Create a size standard as explained in Create a size standard on page 43 Define custom views of analysis tables Refer to the GeneMapper ID Software Versions 3 1 and 3 2 Human Identification Analysis Tutorial Part no 4335523 for more information Define custom views of plots Refer to the GeneMapper ID Software Versions 3 1 and 3 2 Human Identification Analysis Tutorial Part no 4335523 for more information To import the latest panel and bin set from the our web site into the GeneMapper ID Software v3 2 1 database 1 Download and open the file containing panels and bins a Goto www lifetechnologies com support Software Patches amp Updates GeneMapper ID Softwa
12. Save Cancel In the Bin Set field select the AmpFLSTR Bins v2X bin set and configure the stutter distance parameters as shown GeneMapper ID X Software v1 0 1 or higher allows you to specify 4 types of marker repeat motifs tri tetra penta and hexa You can enter parameter values for each type of repeat in the appropriate column Specify the stutter ratio To apply the stutter ratios listed in the Allele tab for single source data deselect the Use marker specific stutter ratio if available check box selected by default Perform appropriate internal validation studies to determine the appropriate filter setting to use Note Applying global stutter ratios may reduce the editing required for single source sample data To apply the stutter ratios contained in the AmpFLSTR Stutter v2X file select the Use marker specific stutter ratio if available check box selected by default Perform appropriate internal validation studies to determine the appropriate filter setting to use AmpFtSTR9 MiniFiler PCR Amplification Kit User Guide 55 4 Chapter 4 GeneMapper ID X Software Set up GeneMapper ID X Software for data analysis Peak Detector tab settings 56 Analysis Method Editor e General Allele Peak Detector Peak Quality SQ amp GQ Settings Detection Algorithm Advanced Ranges Peak Detection Analysis Sizing Beak Amplitude Thresholds Perform Full
13. Figure 13 shows examples of baseline noise and artifacts in an electropherogram while using the MiniFiler Kit You should consider possible noise and artifacts when interpreting data from the MiniFiler Kit on the Applied Biosystems 3130 Genetic Analyzer Note A high degree of magnification y axis is used in Figure 13 to illustrate these artifacts data produced on capillary electrophoresis instrument platforms Figure 13 Examples of baseline noise and reproducible artifacts in data produced on an Applied Biosystems 3130xl Genetic Analyzer AmpFtSTR MiniFiler PCR Amplification Kit User Guide Section 5 1 Developmental Validation Characterization of loci Another type of PCR artifact occurs when the amount of input DNA exceeds the recommended amount 0 50 to 0 75 ng These artifacts were characterized secondary stutter products D135317 and D21S11 as shown in the bottom example in Figure 13 Their mobility varies with that of the main amplification product Genotyping may result in the detection of these artifacts as off ladder alleles or OL Alleles This occurs if the recommended amount of input DNA is exceeded and off scale data is obtained Characterization of loci SWGDAM guideline 2 1 Nature of the polymorphisms Inheritance Mapping The basic characteristics of a genetic marker must be determined and documented SWGDAM July 2003 This section describe
14. 2 Chapter 2 Perform PCR Amplification using bloodstained FTA cards 24 AmpFtSTR MiniFiler PCR Amplification Kit User Guide Perform Electrophoresis Allelic ladder 26 Section 3 1 3100 3100 Avant 3130 3130xl 27 Set up the 3100 3100 Avant and 3130 3130xl instruments for electrophoresis 27 Prepare samples for electrophoresis on the 3100 3100 Avant 3130 3130xl Instruments s RI rp eR I eadeni ect 28 Section 3 2 3500 3500xL instruments 29 Set up the 3500 3500xL instruments for electrophoresis 29 Prepare samples for electrophoresis on the 3500 3500xL instruments 29 Section 3 3 310 Instrument 24 0342544 eee cents 31 Set up the 310 instrument for electrophoresis 31 Prepare samples for electrophoresis on the 310 instrument 31 AmpFtSTR MiniFiler PCR Amplification Kit User Guide 25 Chapter 3 Perform Electrophoresis Allelic ladder requirements Allelic ladder requirements To accurately genotype samples you must run an allelic ladder sample along with the unknown samples Number of 4 din Number of samples per allelic Instrument allelic ladders injection ladder s to run equals 3100 Avant or
15. 24 25 26 27 28 29 0165539 018551 CSF1PO FGA ESReterence Samples 118 124 130 136 142 148 154 160 166 172 178 0251338 lt o 9 5 90 E 9 Click Apply then OK to add the panel and bin set to the GeneMapper ID Software database IMPORTANT If you close the Panel Manager without clicking OK the panels and bins are not imported into the GeneMapper ID Software database AmpFtSTR9 MiniFiler PCR Amplification Kit User Guide 37 4 Chapter 4 Analyze Data Set up GeneMapper ID Software for data analysis Create an analysis Use the following procedure to create an analysis method method 1 Select Tools GeneMapper Manager to open the GeneMapper Manager GeneMapper Manager Last Saved Owner Instrument Analysis Type Description IdentifilerDirect_HID_v1 2010 05 05 10 24 1 gmid HID Default Identifiler 0 Identifiler_Plus_AnalysisMet 2011 05 19 14 41 1 HID Default analysis Microsatellite Default 2010 01 27 14 58 0 grid Microsatellite Factory Provided Done 2 Select the Analysis Methods tab then click New to open the New Analysis Method dialog box 3 Select HID and click OK to open the Analysis Method Editor with the General tab selected 4 Enter the settings shown in the figures on the following pages Note The Analysis Method Editor closes when you save your settings To complete th
16. 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 MiniFiler 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 MiniFiler Allelic Ladder Or An allele that does correspond to an Allelic Ladder allele but whose size is just outside a window because of measurement error 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 Table 3 on page 70 shows typical precision results obtained from five runs 16 capillaries run of the AmpF STR MiniFiler Allelic Ladder on an Applied Biosystems 3130x Genetic Analyzer 36 cm capillary and POP 4 polymer using the 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 sm
17. settings Analysis Method Editor General Allele Peak Detector Peak Quality 150 amp GQ Settings Quality weights are between and 1 Sample and Control GQ Weighting Broad Peak BD Allele Number Out of Bin Allele BIN Low Peak Height LPH Overlap OVL Peak Height MPH Marker Spike SP m Off scale OS Peak Height Ratio PHR Control Concordance CC Weight 1 0 Only applicable to controls r5Q Weighting Broad Peak BD os Allelic Ladder GQ Weighting Spike SSPK SPK Off scale 05 50 amp GQ Ranges Sizing Quality From 0 75 to 1 0 From to 0 25 Genotype Quality From 0 75 to 1 0 From to 0 25 IMPORTANT The values shown are the software defaults and are the values we used during developmental validation Perform appropriate internal validation studies to determine the appropriate values to use Create size The _ 5_ 5500 75 450 size standard definition is installed with the software for standard optional use with the MiniFiler Kit and contains the following size standard peaks GeneScan 500 LIZ Size Standard peak GeneScan 600 LIZ Size Standard v2 0 sizes peak sizes 75 100 139 150 160 200 300 350 400 and 80 100 114 120 140 160 180 200 214 220 450 240 250 260 280 300 314 320 340 360 380 400 414 420 440 and 460 Note The 250 nt and the 340 nt peaks in the
18. 5 5001179 Size Standard The samples were analyzed using both sizing methods and their allele calls were compared The size standard definitions for both methods include all the peaks from 75 to 450 nt with the exception of the 250 nt peak The genotyping results n 36 000 alleles for the two methods were compared for concordance The genotyping accuracy rates for the Local Southern and 3rd Order Least Squares algorithms were equivalent No alleles were labeled with an incorrect genotype and only a very small percentage Local Southern 0 0576 3rd Order Least Squares 0 00876 of the alleles were designated as off ladder when they did not represent a true micovariant allele All of the discordant off ladder allele calls were within 0 08 nt of the 0 5 nt off set for the bin sizing window AmpFtSTR MiniFiler PCR Amplification Kit User Guide Ordering Information Equipment and materials not included Table 15 Equipment Equipment Source Applied Biosystems 3100 3100 Avant Genetic Analyzer Applied Biosystems 3500 3500xL Genetic Analyzer Applied Biosystems 3130 3130xl Genetic Analyzer Contact your local Life Technologies sales representative Veriti 96 Well Thermal Cycler 4375786 GeneAmp PCR System 9700 with the Silver 96 Well Block N8050001 GeneAmp PCR System 9700 with the Gold plated Silver 96 Well Block 4314878 Silver 96 W
19. MiniFiler PCR Amplification Kit User Guide 131 Documentation and Support Obtain SDSs Portable document format PDF versions of this guide and the documents listed above are available at www lifetechnologies com Note To open the user documentation available from the our web site use the Adobe Acrobat Reader software available from www adobe com Obtain SDSs Safety Data Sheets 5055 are available from www lifetechnologies com support Note For the SDSs of chemicals not distributed by Life Technologies contact the chemical manufacturer Obtain support For HID support North America Send an email to HIDTechSupport lifetech com or call 888 821 4443 option 1 Outside North America Contact your local support office For the latest services and support information for all locations go to www lifetechnologies com At the website you can e Access worldwide telephone and fax numbers to contact Technical Support and Sales facilities Search through frequently asked questions FAQs Submit a question directly to Technical Support Search for user documents 51055 vector maps and sequences application notes formulations handbooks certificates of analysis citations and other product support documents Obtain information about customer training Download software updates and patches Limited Product Warranty Life Technologies Corporation and or its affiliate s warrant their prod
20. SWGDAM Guideline 2 2 82 SerisitiVIty mmBmB E A E 83 SWGDAM guideline 2 3 205 adil ka did a add chee deat 83 Effect of DNA quantity on results 83 SINE Ex 84 SWGDAM guideline 2 4 84 Degraded DNA css sa min odd epe eec 9 ies i uei o ipe me lv qwe IY 84 Effect of inhibitors 86 Effect of inhibitors Humic 88 Mixt re St dles5 te Ene e tbc el bote e tentaret teles 90 SWGDAM guideline 2 8 90 AmpFtSTR MiniFiler PCR Amplification Kit User Guide 5 Contents Mixt re scade Kad a kita laya thas tape ius ee ag a K dades 90 Resolution of genotypes in mixed samples 91 Limit of detection of the minor component 92 Population da tarsi tates sie eo eo ee A IUE AL 93 SWGDAM guideline 2 7 tr day Wad kund Weha I EB NE bed Kak belaya b 93 OVerVIBWa set manete Po ndi fo ho nr ft t ef 93 Concordance studies
21. Software Patches amp Updates GeneMapper ID X Software to determine if newer files are available 3 If updated files are available download and import the files into the GeneMapper ID X Software as explained in Import panels bins and marker stutter on page 48 Note When downloading new versions of analysis files refer to the associated Read Me file for details of changes between software file versions If you have validated previous file versions for data analysis conduct the appropriate internal verification studies before using new file versions for operational analysis 4 Create an analysis method as explained in Create an analysis method on page 53 5 Define custom views of analysis tables Refer to Chapter 1 of the GeneMapper ID X Software Version 1 0 Getting Started Guide Pub no 4375574 for more information 6 Define custom views of plots Refer to Chapter 1 of the GeneMapper ID X Software Version 1 0 Getting Started Guide Pub no 4375574 for more information Check panel bin 1 Start the GeneMapper ID X Software then log in with the appropriate user and stutter file name and password version IMPORTANT For logon instructions refer to the GeneMapper ID X Software Version 1 0 Getting Started Guide Pub no 4375574 2 Select Tools Panel Manager AmpFtSTR MiniFiler PCR Amplification Kit User Guide 47 4 Chapter 4 GeneMapper ID X Software Set up GeneMappe
22. Walton A and Gill P 1992 A further tetranucleotide repeat polymorphism in the vWF gene Hum Mol Genet 1 287 Kwok S and Higuchi 1989 Avoiding false positives with PCR Nature 339 237 238 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 H Schmidt L Wei M H Hustad T Leman M L Zbar and Tory K 1993 Three tetranucleotide polymorphisms for loci D351352 0351358 0351359 Hum Mol Genet 2 1327 Magnuson V L Ally D S Nylund S J Karanjawala Z E Rayman J B Knapp J L Lowe A L Ghosh S Collins F S 1996 Substrate nucleotide determined non templated addition of adenine by Taq DNA polymerase implications for PCR based genotyping and cloning Biotechniques 21 700 709 Mansfield E S Robertson J M Vainer M Isenberg A R Frazier R R Ferguson K Chow S Harris D W Barker D L Gill 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 Murray J C 1992 Tetranucleotide repeat polymorphism at the human alpha fibrinogen locus FGA Hum Mol Genet 1 779 AmpFtSTR MiniFiler PCR Amplification Kit U
23. e D 3 D 5 Chapter 5 Performance Validation After Buffer and Enzyme Component Replacement Inhibition study Figure 31 Degraded DNA study representative electropherograms from 500 pg input DNA amplifications of pristine DNA samples for comparison to simulated degraded samples Y scale 5000 RFU 5000 4000 5000 4000 non 2000 D m Control A 2 udal e Test A Test i LL Alu dia LA 65 85 105 125 145 165 185 205 225 245 C Mark Sample for Deletion 65 85 125 165 185 205 225 245 ad l n 65 55 105 125 145 165 185 205 225 245 Control Mark Sample for Deletion 7 Mark Sample for Deletion 65 5 105 125 185 205 225 245 s000 4000 ooo 2000 Test C Mark Sample for Deletion 65 55 105 125 145 165 185 205 225 245 Inhibition study An inhibition series of 0 5 ng control 007 consisting of uninhibited control humic acid at a final concentration of 50 ng uL and hematin at final concentration of 45 uM in replicates of five were amplified using each of Test and Control mixes The amount of each inhibitor tested was titrated to cause an approximate 50 reduction in overall peak height of the samples Results were evaluated for mean peak height minimum peak height intracolor balance and levels of allelic dropout Mean peak height No significant difference in mean peak height or
24. 1 GeneMapper ID Software Set up GeneMapper ID Software for data analysis General Allele Peak Detector Peak Quality 50 amp GQ Settings Min Max Peak Height LPH MPH Homozygous min peak height Heterozygous min peak height Max Peak Height MPH Peak Height Ratio PHR Min peak height ratio Broad Peak BD Max peak width basepairs Allele Number Max expected alleles Allelic Ladder Spike Spike Detection Cut off Value Perform internal validation Enable 0 2 studies to determine settings Factory Defaults IMPORTANT Perform the appropriate internal validation studies to determine the minimum heterozygous and homozygous minimum peak height thresholds and the minimum peak height ratio threshold that allow for reliable interpretation of data 41 lt 97 o 9 5 90 E 4 Chapter 4 Analyze Data Set up GeneMapper ID Software for data analysis Quality Flags tab Analysis Method Editor HID settings General Allele Peak Detector Peak Quality Quality Flags Quality weights are between and 1 gt Quality Flag Settings Spectral Pull up 8 Control Concordance Broad Peak Low Peak Height Out of Bin Allele 08 Off scale Peak Height Ratio Overlap Thresholds Sizing Quality From 075 to10 From 0 0 t
25. 117 lists the required materials not supplied with this kit Reagents and parts IMPORTANT The fluorescent dyes attached to the primers are light sensitive Protect the primer set amplified DNA allelic ladder and size standard from light when not in use Keep freeze thaw cycles to a minimum Electrophoresis software setup and the documents listed in the table The following table lists data collection software and the run modules that can be used to analyze PCR products generated by this kit For details on the procedures refer to reference documents Data ida Collection Bparseng Run modules and conditions References Analyzer System Software Applied 1 0 Windows GeneScan36Avb DyeSetG5Module 3100 3100 Avant Genetic Analyzers Biosystems NT Injection condition 3 kV 5sec Protocols for Processing AmpFtSTR 3100 Avant e GS600v2 0Analvsis gs PCR Amplification Kit PCR Products User Bulletin Part 4332345 Applied 2 0 Windows e HIDFragmentAnalysis36_POP4_1 3100 3100 Avant Genetic Analyzers Biosystems 2000 Injection condition 3kV 10 sec Using Data Collection Software v2 0 3100 e Dve Set G5 Protocols for Processing 5 y PCR Amplification Kit PCR Products User Bulletin Part no 4350218 1 1 Windows GeneScan36vb DyeSetG5Module 3100 3100 Avant Genetic Analyzers NT Injection condition 3kV 10 sec Protocols for Processing AmpFtSTR PCR Amplification Kit PCR Products e G
26. 135 137 139 141 143 0135317 lt Marker 0139317 90 00 139 00 9 Import AmpFLSTR_Stutter_v2X a Select the AmpFLSTR_Panels_v2X folder in the navigation panel Panel Manager File Edit Bins Help i x B BI HB Bin Set AmpFLSTR_Bins_ BI BBE Panel Hame Ofiler v1 1X null e 5 Plus v1 1X null 5 v2 1X Identifiler_Plus_v1 1 null i az Panel Manager CO AmpFLSTR_NGMSElect_v2X AmpFLSTR_NGM_v3x ED AmpFLSTR_Panels_v1X b Select File Import Marker Stutter to open the Import Marker Stutter dialog box c Navigate to then open the AmpFLSTR Analysis Files GMIDX folder AmpFtSTR MiniFiler PCR Amplification Kit User Guide 51 4 Chapter 4 GeneMapper ID X Software Set up GeneMapper ID X Software for data analysis d Select AmpFLSTR Stutter v2X then click Import Note Importing this file associates the marker stutter ratio with the bin set in the AmpFLSTR Panels v2X folder Import Marker Stutter Look in u AmpFLSTR Analysis Files GMIDX E AmpFLSTR_Bins_v2x ampFistR_Panels_v2x My Recent Bi AmpFLSTR Stutter v2X Documents ReadMe AmpFLSTR 2 My Documents File name AmpFLSTR_Stutter_v2X txt My Computer Files of type all Files 10 View the imported marker stutters in the navigation pane a Double click th
27. 180 190 200 210 220 230 240 250 260 270 280 230 29 cycles 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 70 80 90 100 110 120 30 cycles 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 280 31 cycles 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 32 2000 Accuracy precision and reproducibility SWGDAM guideline The extent to which a given set of measurements of the same sample agree with their 29 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 MiniFiler Kit profiles have been determined from various sample types Figure 7 shows the size differences that are typically observed between sample alleles and allelic ladder alleles on the Applied Biosystems 3130 1 Genetic Analyzer with POP 4 polymer The x axis in Figure 7 represents the nominal nucleotide sizes for the AmpF STR MiniFiler Allelic Ladder The dashed lines parallel to the x axis represent 0 25 nt windows The y axis re
28. 2 weeks gt 2 weeks IMPORTANT Store the amplified products so that they are protected from light Amplification using bloodstained FTA cards 22 FTA cards can be useful for collecting storing and processing biological samples A small punch disc of the card containing the sample can be placed directly into an amplification tube purified and amplified without transferring the disc Our studies indicate that a 1 2 mm bloodstained disc contains approximately 5 to 20 ng DNA An appropriate cycle number for this high quantity of DNA is 24 cycles as determined by our validation studies However it is recommended that each laboratory determine the optimum cycle number based on internal validation studies In the example shown in Figure 3 a 1 2 mm disc of a bloodstained FTA card was purified using three washes with FTA Purification Reagent and two washes with 1X low TE buffer The purified punch disc was then amplified in the MicroAmp tube for 24 cycles AmpFtSTR MiniFiler PCR Amplification Kit User Guide 2 2 Amplification using bloodstained FTA cards Figure 3 MiniFiler Kit results from 1 2 mm FTA bloodstain disc 24 cycle amplification analyzed on the Applied Biosystems 3130xl Genetic Analyzer 70 80 30 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 AmpFtSTR MiniFiler PCR Amplification Kit User Guide 23
29. 21 documentation related 131 E electrophoresis Data Collection Software 27 29 31 prepare samples 28 29 preparing samples on the 310 instrument 31 reagents and parts 31 references 27 29 31 run module 27 29 31 set up 31 emission spectra 16 enzyme new 98 equipment not included with kit 117 extra peaks causes 75 F five dye fluorescent system 12 fluorescent dyes 15 cards amplification 22 bloodstained 22 133 Index G GeneMapper ID Software analyze project 44 create analysis method 38 create size standard 43 examine and edit project 45 import panels and bins 34 overview 33 setup 34 GeneMapper ID X Software analyze project 60 check version of panels bins and stutter 47 create analysis method 53 create size standard 58 examine and edit project 61 import panels bins and stutter 48 overview 46 setup 47 size standard create 58 GeneScan size standard about 17 dye label 15 volume per reaction 28 29 31 H Hi Di formamide volume per reaction 28 29 31 identity probability of 95 import size standard 58 inheritance 81 instrumentation 310 genetic analyzer 31 3100 3100 Avant 27 3130 3130 1 27 3500 3500xL 29 software compatibility 15 kit contents 16 description 11 fluorescent dyes 15 instruments for use with 11 loci amplified 12 master mix 16 134 PCR reaction mix 21 primers 11 16 20 purpose 11 reagents 16 storage 16 thermal cyclers for use with 122 kit perfor
30. 240 250 260 tt tt tt 270 280 290 70 80 70 80 30 30 100 100 110 110 120 120 130 130 140 140 150 150 160 160 170 170 180 180 190 190 200 200 210 210 220 230 230 250 250 260 260 270 270 280 280 290 290 MiniFiler Kit reactions were amplified for 28 29 30 31 and 32 cycles on the Silver 96 Well GeneAmp PCR System 9700 using 0 50 ng from three DNA samples As expected the amount of PCR product increased with the number of cycles A full profile was generated 28 cycles and off scale data were collected for several allele peaks at 32 cycles Figure 6 PCR cycle number While none of the cycle numbers tested produced nonspecific peaks 30 cycles was found to give optimal sensitivity when the amplified products were examined on Applied Biosystems 3130x Genetic Analyzers AmpFtSTR9 MiniFiler PCR Amplification Kit User Guide 67 Chapter 5 Experiments and Results Accuracy precision and reproducibility Figure 6 Representative MiniFiler Kit profiles obtained from amplification of 0 50 ng DNA template using 28 29 30 31 32 cycles analyzed on an Applied Biosystems 3130xl Genetic Analyzer y axis 4000 RFU anoo 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 230 28 cycles 2000 0 70 80 90 100 110 120 130 140 150 160 170
31. 3130 Genetic Analyzer AmpFtSTR9 MiniFiler PCR Amplification Kit User Guide 83 Chapter 5 Experiments and Results Stability Stability SWGDAM guideline 2 4 Degraded DNA 84 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 GWGDAM July 2003 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 nanogram of degraded DNA was amplified using the MiniFiler Kit and Identifiler Kit Two nanograms of degraded DNA was amplified usin
32. 3130 1 per 4 injections 4 samples 15 samples 1 allelic ladder 3100 or 3130 1 per injection 16samples 15 samples 1 allelic ladder 3500 1 per 3 injections 8 samples 23 samples 1 allelic ladder 3500xL 1 per injection 24 samples 23 samples 1 allelic ladder IMPORTANT Variation in laboratory temperature can cause changes in fragment migration speed and sizing variation between both single and multiple capillary runs with larger size variations seen between samples injected in multiple capillary runs We recommend 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 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 26 AmpFtSTR MiniFiler PCR Amplification Kit User Guide Section 3 1 3100 3100 Avant and 3130 3130xl instruments Set up the 3100 3100 Avant and 3130 3130xl instruments for electrophoresis Section 3 1 3100 3100 Avant and 3130 3130xl instruments Set up the 3100 3100 Avant and 3130 3130xl instruments for electrophoresis Appendix C Ordering Information on page
33. 65 Developmental 65 Accuracy precision and reproducibility 68 Extra peaks in the 75 Characterization 2 81 Species specificitys Js veu k ea Sakae baka Fees Wik k Se ee 82 OTST VIC eos RD toc tente cd 83 Stability eie cts be sl ka 84 Mixture studies bed de en E en 90 Population data o eee ee EN ee ees 93 Mutation ss sn d s la ser d seb mad y d We y W y I bul eee 95 Probability of identity 95 Probability of paternity exclusion 96 Section 5 2 Performance Validation After Buffer and Enzyme Component Replacement Dee ekle euim is em v nn 98 OVeEVIeW Ju dd eder eed ees at te Gta deed get edm 98 Experiments vina b aon epe o otn ae A sm e e ton 98 Reproducibility study 99 Sensitivity SUAV 101 Degraded DNA st dy i 4 43 8 e e re baki kk 105 hibition stidyo multe md 108 112 AmpFtSTR MiniFiler P
34. A nucleotide addition 78 standards for samples 17 STRBase 94 stutter check version 47 import 48 stutter peak or products 75 stutter percentages marker specific 78 support obtaining 132 AmpFtSTR MiniFiler PCR Amplification Kit User Guide Index T technical support 132 Terms and Conditions 132 thermal cyclers for use with kit 122 programming conditions 22 training information on 132 troubleshooting 113 U user supplied reagents 19 validation accuracy precision reproducibility 68 annealing temperatures 66 67 characterization of loci 81 conditions 65 developmental 65 effect of DNA quantity 83 loci characterization 81 mutation rate 95 PCR cycle number 67 performance after buffer and enzyme replacement 98 population data 93 probability of identity 95 probability of paternity exclusion 96 sensitivity 83 size deviation sample and ladder alleles 68 69 species specificity 82 stutter 75 thermal cycling parameters 66 W warranty 132 work area amplified DNA 122 PCRsetup 121 setup and lab design 121 workflow overview 14 135 9 Headquarters 2 5791 Van Allen Carlsbad 92008 USA Phone 1 760 603 7200 Free USA 800 955 6288 Bn For support visit www appliedbiosystems com support technologies www lifetechnologies com
35. AmpFLSTR_MiniFiler_GS500_Panels_v1 tot My Documents Files of type All Files 6 Import the bins file Panel Manager File Edit Bins View P AmpFLSTR_MiniFiler Panels v 8 L7MiniFiler GS500 v1 a Select the AmpFISTR MiniFiler GS500 Panels v1 folder in the navigation pane b Select File Import Bin Set to open the Import Bin Set dialog box c Navigate to then open the MiniFiler Analysis Files GMID folder AmpFtSTR9 MiniFiler PCR Amplification Kit User Guide 35 4 Chapter 4 Analyze Data Set up GeneMapper ID Software for data analysis 7 View the imported panels in the navigation pane Panel Manager File Edit Bins View d Select AmpFLSTR MiniFiler GS500 Bins vl1 txt then click Import Note Importing this file associates the bin set with the panels in the AmpFISTR MiniFiler GS500 Panels v1 folder Import Bin My Recer t Documents My Documents Set MiniFiler Analysis Files GMID E AmpFLSTR E AmpFLSTR MiniFiler GS500 Panels 1 154 55500 v1 xml File name Files of type AmpFLSTR_MiniFiler_GSSO0_Bins_v1 tt Al Files a Double click the AmpFISTR_MiniFiler_GS500_Panels_v1 folder to view the MiniFiler GS500 v1 folder b Double click the MiniFiler GS500 v1 folder to display the panel information in the right pane mmm B n S
36. 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 Int J Legal Med 109 186 194 Sparkes R Kimpton C Gilbard S Carne P Andersen J Oldroyd N Thomas D Urquhart A and Gill P 1996b The validation 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 1993 A microsatellite genetic linkage map of human chromosome 18 Genomics 15 48 56 Szibor Lautsch S Plate I Bender and Krause D 1998 Population genetic data of the STR HumD351358 in two regions of Germany Intl J Legal Med 111 160 161 Wallin J M Buoncristiani M R Lazaruk K D Fildes N Holt C L Walsh P S 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 PS 2002 Constructing universal multiplex PCR systems for comparative genotyping J 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 vWA Nucleic Acids Res 24 2807 2812 Webe
37. GeneScan 500 LIZ Size Standard are not included in the size standard definition These peaks can be used as an indicator of precision within a run Use the following procedure if you want to create your own size standard 1 Select Tools GeneMapper Manager to open the GeneMapper Manager 58 AmpFtSTR MiniFiler PCR Amplification Kit User Guide Section 4 2 Software 4 Set up GeneMapper ID X Software for data analysis 2 Select the Size Standards tab then click New GeneMapper ID X Manager Find Name Containing STE Projects Analysis Methods Table Settings Plot Settings Matrices Size Standards Report Settings Name Last Saved Owner Type Description CE F HID GS500 75 400 2007 08 09 13 23 gmidx Advanced 65500 75 450 2007 08 09 13 24 gmidx Advanced CE G5 HID GS500 2006 10 11 13 12 2 gmidx Advanced 45600 LIZ 2007 06 26 10 43 1 gmidx Advanced GS600 LIZ Normalization 80 400 2007 06 27 01 43 1 gmidx Advanced 95600 LIZ 80 400 2007 06 27 01 43 1 gmidx Advanced Open Export Delete C gt lt o 19 o e e P 122 E S 3 Enter a In the Size Standard Dye field select Orange In the Size Standard Table enter the sizes specified in on page 58 The example below is for the GeneScan 500 LIZ Size Standard Size Standard Editor E
38. Kimura K 1994 Identification of the heme compound copurified with deoxyribonucleic acid DNA from bloodstains a major inhibitor of polymerase chain reaction PCR amplification J Forensic Sci 39 362 372 Barber M D and Parkin B H 1996 Sequence analysis and allelic designation of the two short tandem repeat 018551 and 0851179 Intl J Legal Med 109 62 65 Baron H Fung S Aydin A Bahrig S Luft F C Schuster H 1996 Oligonucleotide ligation assay OLA for the diagnosis of familial hypercholesterolemia Nat Biotechnol 14 1279 1282 Begovich A B McClure G R Suraj V C Helmuth Fildes N Bugawan T L Erlich H A Klitz W 1992 Polymorphism recombination and linkage disequilibrium within the HLA class II region J Immunol 148 249 58 Bender K Farfan M J Schneider 2004 Preparation of degraded human DNA under controlled conditions Forensic Sci Int 139 134 140 Brinkman B Klintschar M Neuhuber F Huhne J and Rolf B 1998 Mutation rate in human microsatellites Influence of the structure and length of the tandem repeat Am J Hum Genet 62 1408 1415 Brinkman B Moller A and Wiegand P 1995 Structure of new mutations in 2 STR systems Intl J Legal Med 107 201 203 Butler J M 2005 Forensic DNA Typing Burlington MA Elsevier Academic Press Butler J M Shen Y McCord 2003 The development of reduced size STR amplicons as tools for anal
39. Quantifying the amount of DNA in a sample before amplification allows you to quantification determine whether or not sufficient DNA is present to permit amplification and to calculate the optimum amount of DNA to add to the reaction The optimum amount of DNA for the Identifiler Kit is 1 0 ng in a maximum input volume of 10 uL for 28 PCR cycles AmpFtSTR MiniFiler PCR Amplification Kit User Guide 19 Chapter 2 Perform DNA quantification If too much DNA is added to the PCR reaction then the increased amount of PCR product that is generated can result in e 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 is 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 and it results 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 Methods of quantifying DNA Life Technologies provides several kits for quantifying DNA in samples See the references cited in the following table for details about these kits Pr
40. Sample 11 fsa Sample MiniFiler_AnalysisMetho MiniFiler GS500 v1 Sample 12 fsa Sample MiniFiler amp nalysisMetho MiniFiler 55500 v1 Sample 13 fsa Sample MiniFiler_AnalysisMetho MiniFiler 55500 v1 Sample 14 fsa Sample MiniFiler amp nalysisMetho MiniFiler GS500 v1 of w ol al Sample 15 fsa Sample MiniFiler _AnalysisMetho MiniFiler GS500 v1 Sample 15 fsa Sample MiniFiler amp nalysisMetho MiniFiler 55500 v1 Sample 17 Sample MiniFiler amp nalysisMetho MiniFiler GS500 v1 Sample 18 fsa Positive Control MiniFiler amp nalysisMetho MiniFiler GS500 v1 Sample 19 fsa Negative Contrc MiniFiler_AnalysisMetho MiniFiler GS500 v1 Sample 2 fsa Sample MiniFiler amp nalysisMetho MiniFiler 55500 v1 Sample 3 fsa Sample MiniFiler amp nalysisMetho MiniFiler GS500 v1 Sample 4 fsa Sample MiniFiler amp nalysisMetho MiniFiler 55500 v1 Sample 5 fsa Sample MiniFiler_AnalysisMetho MiniFiler GS500 v1 Sample 6 fsa Sample MiniFiler amp nalysisMetho MiniFiler 55500 v1 Sample 7 fsa Sample MiniFiler amp nalysisMetho MiniFiler GS500 1 Sample 8 fsa Sample MiniFiler amp nalysisMetho MiniFiler GS500 v1 A Ak A A Ae Ak A Ak Ak Aie Ak Aue Ak A Aie 4 Sample 9 fsa Sample MiniFiler amp nalysisMe
41. Total 0 0 0 Sample Quality per project based on sample PQVs SOS SSPK MIX OMR SQ CGQ Total of Samples 8 thresholds met more thresholds not met Samples 47 4l 6 gt Analysis Completed Examine and edit a project You can display electropherogram plots from the Samples and Genotypes tabs of the Project window to examine the data These procedures start with the Analysis Summary tab of the Project window assuming the analysis is complete AmpFtSTR9 MiniFiler PCR Amplification Kit User Guide 61 Chapter 4 GeneMapper ID X Software For more information For more information For more information refer to e GeneMapper ID X Software Version 1 0 Getting Started Guide Pub no 4375574 e GeneMapper ID X Software Version 1 0 Quick Reference Guide Pub no 4375670 GeneMapper ID X Software Version 1 0 Reference Guide Pub no 4375671 GeneMapper ID X Software Version 1 1 Mixture Analysis Getting Started Guide Pub no 4396773 e GeneMapper ID X Software Version 1 2 Reference Guide Pub no 4426481 e GeneMapper ID X Software Version 1 2 Quick Reference Guide Pub no 4426482 62 AmpFtSTR MiniFiler PCR Amplification Kit User Guide Experiments Results Section 5 1 Developmental Validation 65 Aida esu ene eeu eee ion ee nete e E des
42. amounts using standard PCR and capillary electrophoresis conditions for the MiniFiler Kit Primates gorilla chimpanzee orangutan and macaque 0 50 ng each Non primates mouse dog pig cat horse hamster rat chicken and cow 10 ng each e Microorganisms Candida alb icans Escherichia coli Lactob acillus casei Staphylococcus aureus Neisseria gonorrhoeae Bacillus sub tilis and Lactob acillus rhamnosus equivalent to 10 copies The chimpanzee and gorilla DNA samples produced partial profiles within the 70 to 283 nucleotide region The remaining species tested did not yield reproducible detectable products Figure 14 on page 82 shows example electropherogram results from the species specificity tests Figure 14 Representative electropherograms for some species tested in a species specificity study including positive and non template controls NTC 82 270 Human Control DNA 007 290 290 260 270 280 Microbial Pool 260 270 280 290 Negative Control AmpFtSTR MiniFiler PCR Amplification Kit User Guide Section 5 1 Developmental Validation Sensitivity Sensitivity SWGDAM guideline When appropriate the range of DNA quantities able to produce reliable typing 23 results should be determined SWGDAM July 2003 Effect of DNA If too much DNA is added to the PCR reaction the increased amount of PCR product quantity on results that is generated can result in Fluoresc
43. 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 In the U S U S Department of Health and Human Services guidelines published in Biosafety in Microbiological and Biomedical Laboratories found at www cdc gov biosafety Occupational Safety and Health Standards Bloodborne Pathogens 29 CFR 1910 1030 found at 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 In the EU Check local guidelines and legislation on biohazard and biosafety precaution and refer to the best practices published in the World Health Organization WHO Laboratory Biosafety Manual third edition found at www who int csr resources publications biosafety WHO CDS CSR LYO 2004 11 AmpFtSTR MiniFiler PCR Amplification Kit User Guide 125 Appendix E Safety Biological hazard safety 126 AmpFtSTR MiniFiler PCR Amplification Kit User Guide Bibliography Akane A Matsubara K Nakamura H Takahashi S and
44. d an ela Genetic Analyzer Genetic Analyzer Genetic Analyzer Analyze 2 RET data JN j GeneMapper D X or GeneMapper D Software 14 AmpFtSTRO MiniFiler PCR Amplification Kit User Guide Chapter 1 Overview 1 Instrument and software overview Instrument and software overview Data collection and analysis software Instrument and software compatibility About multicomponent analysis How multicomponent analysis works This section provides information about the data collection and analysis software versions required to run the this kit on specific instruments The data collection software provides instructions to firmware running on the instrument and displays instrument status and raw data in real time As the instrument measures sample fluorescence with its detection system the data collection software collects the data and stores it The data collection software stores information about each sample a sample file fsa files for 31xx instruments and hid files for 3500 instruments which is then analyzed by the analysis software Instrument Operating system Data co recton Analysis software software 3100 3100 Windows NT 1 1 3100 GeneMapper ID Avant 1 0 3100 Avant Software v3 2 1 T e GeneMapper D X Windows 2000 2 0 Software v1 0 1 or higher 3130 3130xIt Windows XP 3 0 3500 3500xL Windows XP 3500 Series Data GeneMapper D X Soft
45. different instruments of the same platform type because of these factors We strongly recommend that the allele sizes be compared to the sizes obtained for known alleles the AmpF STR MiniFiler Allelic Ladder from the same run and then be converted to genotypes as described in Before you start on page 33 GeneMapper ID Software and Before you start on page 46 GeneMapper ID X Software See Table 3 for the results of five runs of the AmpF STR MiniFiler Allelic Ladder an Applied Biosystems 3130 Genetic Analyzer For more information on precision and genotyping see Lazaruk et al 1998 and Mansfield et al 1998 In Table 3 the mean sizes for all the alleles in each run 16 capillaries were calculated The mean range shown in the table represents the lowest and highest mean size values obtained across all 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 3 represents the lowest and highest standard deviation values obtained across all five runs Table 3 Precision results of five runs 16 capillaries run of the AmpF STR MiniFiler Allelic Ladder Allele Mean Standard Deviation AMEL X 101 54 101 59 0 024 0 037 107 51 107 56 0 029 0 038 CSF1PO 6 86 65 86 67 0 027 0 038 7 90 70 90 72 0 026 0 038 8 94 72 94 77 0 023 0 036 9 98 76 98 79 0 033 0 041 10 10
46. federal state and local requirements for container storage After emptying a waste container seal it with the cap provided Characterize by analysis if necessary the waste generated by the particular applications reagents and substrates used in your laboratory Ensure that the waste is stored transferred transported and disposed of according to all local state provincial and or national regulations Radioactive or biohazardous materials may require special handling and disposal limitations may apply ea chemical CAS Chemical Phrase handling 26628 22 8 Sodium Azide Sodium azide may react with lead and copper plumbing to form highly explosive metal azides 124 AmpFtSTR MiniFiler PCR Amplification Kit User Guide Appendix E Safety E Biological hazard safety Biological hazard safety Q WARNING Potential Biohazard Depending on the samples used on this instrument the surface may be considered a biohazard Use appropriate decontamination methods when working with biohazards A WARNING 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 All work should be
47. heights on Life Technologies instruments provides additional valuable data to aid in resolving mixed genotypes This quantitative value is much less subjective than comparing relative intensities of bands on a stained gel AmpFtSTR MiniFiler PCR Amplification Kit User Guide 91 Chapter 5 Experiments and Results Mixture studies Ultimately the likelihood that any sample is a mixture must be determined by the analyst in the context of each particular case including the information provided from known reference sample s Figure 19 Amplification of DNA mixtures at various ratios 1 0 15 1 7 1 3 1 1 1 0 1 Limit of detection Mixtures of two DNA samples were examined at various ratios 0 1 1 1 3 1 7 1 15 1 of the minor 1 0 The total amount of genomic input DNA mixed at each ratio was 1 ng The component samples were amplified a PCR System 9700 and were electrophoresed and detected using an Life Technologies 3130x Genetic analyzer The results of the mixed DNA samples are shown in Figure 19 above where samples A and B were mixed according to the ratios provided 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 mi
48. light when not in use Keep freeze thaw cycles to a minimum Electrophoresis The following table lists data collection software and the run modules that you can use software setup and te analyze PCR products generated by this kit For details on the procedures refer to the documents listed in the table a 2 E 9 a a 0 e 5 reference documents Genetic Data Operatin Collection 9 Run modules conditions References Analyzer System Software Applied 1 0 Windows e HID36_POP4 Applied Biosystems 3500 Biosystems XP Injection conditions 1 2kV 15 sec 3500xL Genetic Analyzer User 3500 Guide Part no 4401661 or Dye Set G5 Applied Windows e HID36_POP4 ee ee j NON Re Analyzers Quick Reference Card Biosystems Vista Injection conditions 1 2kV 24 sec Part no 4401662 3500xL oe Dye Set G5 Prepare samples for electrophoresis on the 3500 3500xL instruments Prepare the samples for electrophoresis immediately before loading 1 Calculate the volume of Hi Di Formamide and GeneScan 600 LIZ Size Standard v2 0 needed to prepare the samples Reagent Volume per reaction GeneScan 600 1129 Size Standard v2 0 0 5 uL Hi Di Formamide 8 5 uL Note Include additional samples in your calculations to provide excess volume for the loss that occurs during reagent transfers IMPORTANT The volume of size standard indica
49. 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 experiments and results Pipet the required volumes of components into an appropriately sized polypropylene tube Vortex the tube then centrifuge briefly Into each well of a MicroAmp Optical 96 Well Reaction Plate add 9uL of the formamide size standard mixture 1uL 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 vortex and centrifuge 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 C Immediately place the plate on ice for 3 minutes Prepare the plate assembly on the autosampler Start the electrophoresis run AmpFtSTR MiniFiler PCR Amplification Kit User Guide Chapter 3 3500 3500xL Instruments 3 Set up the 3500 3500xL instruments for electrophoresis Section 3 2 3500 3500xL instruments Set up the 3500 3500xL instruments for electrophoresis Reagents and parts Appendix Ordering Information on page 117 lists the required materials not supplied with this kit IMPORTANT The fluorescent dyes attached to the primers are light sensitive Protect the primer set amplified DNA allelic ladder and size standard from
50. mean minimum peak height was m peak height and intracolor balance 10 inimum mean observed for any Test or Control mixes tested on Control DNA 007 inhibited with hematin or humic acid A significant increase in intracolor balance was observed only for Control A Mix on Control DNA 007 inhibited with Hematin Figure 32 33 and 34 More variation was seen in mean peak height mean minimum peak height and intracolor balance on uninhibited DNA This is not unexpected because the MiniFiler Kit was designed and developed for use on inhibited or degraded samples and is optimized for performance on such sample types All results obtained for all Test and Control mixes fall within the expected range of performance for the MiniFiler Kit 8 AmpFtSTR MiniFiler PCR Amplification Kit User Guide Section 5 2 Performance Validation After Buffer Enzyme Component Replacement Inhibition study Figure 32 Inhibition study mean referenced peak height Lot Control Control B 9 Test Test 8 Test U D Es 3 3 hy 2 e 0 5 ay 2 00 e ra 2 oH m N lt 3 9 2 9 5 e D 3 D 5 Lot Control Control B 9 Test A A Test 8 p Test C Minimum Referenced Peak Height Panel variable Inhibitor AmpFtSTR MiniFiler PCR Amplification Kit User Guide 109 Chapter 5 Performance
51. receipt AmpF STR MiniFiler Contains amplified alleles 1 tube 0 05 mL 2 to 8 C after initial Allelic Cadde See Table 1 on page 12 for a list of alleles included in the allelic ladder Store protected from light The Control DNA 007 is included at a concentration appropriate to its intended use as an amplification control i e to provide confirmation of the capability of the kit reagents to generate a profile of expected genotype The Control DNA 007 is not designed to be used as a DNA quantitation control and you may see variation from the labelled concentration when quantitating aliquots of the Control DNA 007 16 AmpFtSTR MiniFiler PCR Amplification Kit User Guide Chapter 1 Overview 1 Materials and equipment Standards for For the MiniFiler Kit the panel of standards needed for PCR amplification PCR samples product sizing and genotyping are AmpFE STR Control DNA 007 A positive control for evaluating the efficiency of the amplification step and STR genotyping using the AmpF STR MiniFiler Allelic Ladder e GeneScan 500 LIZ Size Standard or GeneScan 600 LIZ Size Standard v2 0 Used for obtaining sizing results These standards which have been evaluated as internal size standards yield precise sizing results for MiniFiler Kit PCR products Order GeneScan 500 LIZ Size Standard Part no 4322682 or the GeneScan 600 LIZ Size Standard v2 0 Part no 4408399
52. the detection of both total human and male DNA in one PCR reaction The kit detects single stranded and degraded DNA How it works The Quantifiler Duo DNA Quantification Kit consists of target specific and internal control 5 nuclease assays The Quantifiler Duo kit combines two human specific assays in one PCR reaction for total human DNA and human male DNA The two human DNA specific assays each consist of two PCR primers and TagMan probe The TagMan probes for the human DNA and human male DNA assays are labeled with VIC and FAM dyes respectively In addition the kit contains an internal PCR control IPC assay similar in principle to that used in the other Quantifiler kits but labeled with NED dye 20 AmpFtSTR MiniFiler PCR Amplification Kit User Guide Chapter 2 Perform 2 Prepare the amplification kit reactions Prepare the amplification kit reactions 1 Calculate the volume of each component needed to prepare the reactions using the table below DNA sample Volume per reaction AmpF STR MiniFiler Master Mix 10 0 uL AmpF STR MiniFiler Primer Set 5 0 uL Note The volumes indicated above include a slight overfill to account for the loss that occurs during reagent transfers 2 Prepare reagents Thaw the PCR Reaction Mix and the Identifiler Primer Set then vortex all reagent tubes including the enzyme for 3 seconds and centrifuge briefly before opening the tub
53. were found 0135317 1 73 in African Americans 0 57 in Caucasians and 1 45 in Hispanics 075820 0 29 in African Americans CSF1PO 0 48 in Hispanics D165539 1 7396 in African Americans and 0 64 in Asians and 1018551 0 48 in Hispanics The variants leading to discordant genotypes in the D135317 locus have been characterized previously Drabek 2004 AmpFtSTR MiniFiler PCR Amplification Kit User Guide Mutation rate Section 5 1 Developmental Validation Mutation rate Estimating germ Estimation of spontaneous or induced germ line mutation at genetic loci can be line mutations 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 of ten 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 y length based STR mutation was observed at the D18S11 locus mutations were not detected at any of the other nine STR loci D18511 mutation was represented by E increase of one 4 nt repeat unit allele 17 was inherited as allele 18 single step S mutation The maternal paternal source of this mutation could not be distinguished r Q Additional Additional studies Edwards et al 1991 Edwards et al 1992 Weber and Wo
54. 0 11 8 21 x 10 11 1 05 x 10 10 2 08 x 10 10 AmpFtSTR MiniFiler PCR Amplification Kit User Guide 95 Chapter 5 Experiments and Results Probability of paternity exclusion Probability of paternity exclusion Table 11 shows the Probability of paternity exclusion Pg values of the MiniFiler Kit STR loci individually and combined Table 11 Probability of paternity exclusion Pg values for the MiniFiler Kit loci Locus African Caucasian Hispanic Native American American CSF1PO 0 545 0 496 0 450 0 409 0251338 0 748 0 725 0 671 0 399 075820 0 591 0 582 0 574 0 492 0135317 0 383 0 487 0 638 0 370 0165539 0 649 0 566 0 567 0 428 018551 0 760 0 731 0 767 0 329 021511 0 737 0 708 0 586 0 399 FGA 0 760 0 766 0 739 0 309 Combined 0 99985 0 99976 0 99970 0 98188 The Pg value is the probability averaged over all possible mother child pairs that a random alleged father will be excluded from paternity after DNA typing using the MiniFiler Kit STR loci Chakraborty Stivers and Zhong 1996 96 AmpFtSTR MiniFiler PCR Amplification Kit User Guide Section 5 1 Developmental Validation Probability of paternity exclusion g lt e 3 o 5 E S 5 2 AmpFtsTR MiniFiler PCR Amplification Kit User Guide 97 Chapter 5 Performance Validation After Buffer and Enzyme Component Replacement Overview Section 5 2 Performance Validation
55. 0 RFU at the 018551 locus in the NED dye yellow at the FGA locus in the PET dye red Y scale 400 RFU 85 105 125 45 465 185 205 225 400 7 Mark Sample for Deletion 200 7 Mark Sample for Deletion 105 125 145 165 185 205 225 85 400 300 2 Sample for Deletion 85 105 125 445 165 485 205 225 Ll Lo Figure 27 Sensitivity study electropherogram of 125 ng Sample 3 amplified with Test B Mix One allele at the D7S820 locus in the FAM dye blue is below the analysis threshold of 50 RFU Y scale 300 RFU 405 425 145 155 195 205 7 Mark Sample for Deletion O Mark Sample for Deletion 85 105 125 145 165 185 205 25 200 160 120 o e A A er fe Ie e n ca Mark Sample for Deletion e5 405 125 445 165 185 205 225 u o S NES ete a 104 AmpFtSTR MiniFiler PCR Amplification Kit User Guide Section 5 2 Performance Validation After Enzyme Component Replacement Degraded study Genotype Genotypes for Test and Control mixes were 100 concordant Table 13 concordance Table 13 Sensitivity study genotype concordance 70 DNA In
56. 0xl Genetic Analyzers 4352755 3100 3100 Avant Genetic Analyzer Autosampler Plate Kit 96 well 4316471 GeneScan 500 LIZ Size Standard 4322682 OR OR GeneScan 600 LIZ Size Standard v2 0 4408399 Running Buffer 10X 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 3130xl instrument refer to Appendix A of the Applied Biosystems 3130 3130xl Genetic Analyzers Maintenance Troubleshooting and Reference Guide Part no 4352716 3500 3500xL Analyzer materials Anode buffer container 4393927 Cathode buffer container 4408256 DS 33 Matrix Standard Kit Dye Set G5 4345833 POP 49 polymer 960 samples for 3500 3500xL Genetic Analyzers 4393710 POP 49 polymer 384 samples for 3500 3500xL Genetic Analyzers 4393715 GeneScan 600 1179 Size Standard v2 0 4408399 Conditioning reagent 4393718 8 Capillary array 36 cm for 3500 Genetic Analyzers 4404683 24 Capillary array 36 cm for 3500xL Genetic Analyzers 4404687 96 well retainer amp base set Standard 3500 3500xL Genetic Analyzers 4410228 8 Tube retainer amp base set Standard for 3500 3500xL Genetic Analyzers 4410231 8 Strip Septa for 3500 3500xL Genetic Analyzers 4410701 96 Well Septa for 3500 3500xL Genetic Analyzers 4412614 Septa Cathode Buffer Container 3500 series 4410715 For a
57. 115 135 155 175 195 215 235 255 75 3000 5 10007 Mark Sample for Deletion 95 115 135 155 175 195 215 235 255 Test 10004 0 Allelic dropout No allelic dropout events were seen for any Test or Control mixes tested on uninhibited Control DNA 007 and Control DNA 007 inhibited with hematin or humic acid Conclusions 112 Laboratories can expect to obtain equivalent quality profiles across a wide range of forensic samples when using the MiniFiler Kit containing the AmpliTaq Gold enzyme and 10X PCR Buffer II manufactured by Life Technologies as compared to the original MiniFiler Kit containing AmpliTaq Gold enzyme and 10X PCR Buffer manufactured by Roche Molecular Systems AmpFtSTR MiniFiler PCR Amplification Kit User Guide Troubleshooting Follow the actions recommended in this appendix to troubleshoot problems that occur during analysis Table 14 Troubleshooting Observation Possible causes Recommended actions Faint or no signal from both the Control DNA 007 and the DNA test samples at all loci Incorrect volume or absence of Master Mix or Primer Set Repeat amplification No activation of DNA Polymerase Repeat amplification making sure to hold reactions initially at 95 C for 1 minute Master Mix not vortexed thoroughly before aliquoting Vorte
58. 17 0 17 0 14 0 14 0 14 Traces of humic acid may inhibit the PCR amplification of DNA evidence collected from soil In this study we tested increasing amounts of humic acid in the PCR amplification of 1 ng of Control DNA 007 with the Identifiler Kit and the MiniFiler Kit and 2 ng with the SGM Plus Kit As the concentration of humic acid increased in the reaction the larger Identifiler Kit and SGM Plus Kit loci failed to amplify However the MiniFiler Kit loci efficiently amplified the DNA at concentrations of humic acid that inhibited the amplification of DNA with the Identifiler Kit and SGM Plus Kit Figure 18 on page 89 The concentrations of humic acid tested were 0 10 30 and 50 ng uL AmpFtSTR MiniFiler PCR Amplification Kit User Guide Section 5 1 Developmental Validation Stability Figure 18 Amplification of Control DNA 007 in the presence of humic acid analyzed on the Applied Biosystems 3130xl Genetic Analyzer 1 ng DNA 007 Untreated Identifiler Kit 1 ng DNA 007 30 ng uL Humic acid g lt e 3 D e 5 5 E e 2 8888828882228 o 2 ng DNA 007 Untreated SGM Plus Kit E 2 ng DNA 007 30 ng uL Humic acid 1 ng DNA 007 Untreated MiniFiler Kit 1 ng DNA 007 30 ng uL Humic acid Comparison of perf
59. 2 79 102 81 0 028 0 038 11 106 80 106 85 0 031 0 044 12 110 82 110 85 0 030 0 043 13 114 83 114 88 0 027 0 045 AmpFtSTR MiniFiler PCR Amplification Kit User Guide Section 5 1 Developmental Validation Accuracy precision and reproducibility Allele Mean Standard Deviation 14 118 83 118 87 0 023 0 041 15 122 83 122 89 0 031 0 041 0135317 8 103 25 103 28 0 029 0 039 9 107 38 107 43 0 028 0 042 10 111 50 111 54 0 035 0 044 11 115 63 115 66 0 031 0 045 12 119 73 119 78 0 037 0 044 13 123 82 123 85 0 038 0 047 14 127 83 127 88 0 038 0 049 15 131 93 131 97 0 035 0 051 0165539 5 74 96 75 01 0 033 0 047 8 87 58 87 61 0 030 0 044 9 91 78 91 81 0 021 0 039 10 95 91 95 95 0 038 0 046 11 100 06 100 09 0 038 0 046 12 104 20 104 22 0 041 0 045 13 108 30 108 36 0 031 0 044 14 112 42 112 46 0 037 0 050 15 116 52 116 58 0 037 0 048 018551 7 124 68 124 73 0 035 0 060 9 132 53 132 57 0 044 0 059 10 136 46 136 50 0 040 0 056 10 2 138 37 138 42 0 040 0 056 11 140 38 140 43 0 038 0 055 12 144 33 144 37 0 039 0 059 13 148 27 148 31 0 048 0 054 13 2 150 19 150 22 0 040 0 062 14 152 21 152 24 0 043 0 057 14 2 154 14 154 18 0 035 0 054 15 156 17 156 20 0 042 0 061 16 160 13 160 16 0 047 0 060 17 164 06 164 10 0 046 0 057 18 168 05 168 06 0 039 0 058 19 172 00 172 02 0 041 0 054 20 175 97 175 99 0 035 0 061 AmpF
60. 75 and 450 nt or 75 and 400 nt depending on the kit In the MiniFiler Kit the amplicon sizes for the large AmpF STR loci have been reduced to improve genotyping performance with degraded and inhibited DNA samples The allele range for the MiniFiler Kit is 70 to 283 nt In order to use the Local Southern algorithm at least one peak lt 70 nt would have to be detected Although the GeneScan 500 LIZ Size Standard does contain 50 nt and 35 nt size standard peaks they are often difficult or impossible to detect The fragments are obscured by the primer front associated with the MiniFiler Kit amplifications Because fragment sizes cannot be extrapolated when using the Local Southern algorithm we recommend the 3rd Order Least Squares algorithm as an alternative sizing method for the MiniFiler Kit For a full description of the Least Squares Method refer to the GeneMapper ID Software Version 3 1 Human Identification Analysis User Guide Part no 4338775 AmpFtSTR MiniFiler PCR Amplification Kit User Guide 115 Appendix The 3rd Order Least Squares Sizing Method Comparing genotyping accuracy Comparing genotyping accuracy 116 We compared the Local Southern and 3rd Order Least Squares methods for genotyping accuracy using a data set of 1 156 Identifiler Kit amplifications The amplified samples were from a single source electrophoresed on an Applied Biosystems 3100 or 3130xl instruments and sized with the
61. 8 FGA 904 88 0 89 4 53 9 100 0 If an unusually low peak height ratio is observed for one locus and there are no other indications that the sample is a mixture the sample may be reamplified and reanalyzed to determine if the imbalance is reproducible Possible causes of imbalance at a locus are Degraded DNA e Presence of inhibitors Extremely low amounts of input DNA SNP in one of the primer binding sites e Presence of an allele containing a rare sequence that does not amplify as efficiently as the other allele Resolution of A sample containing DNA from two sources can be comprised at a single locus of genotypesin mixed of the seven genotype combinations listed below samples Heterozygote heterozygote no overlapping alleles four peaks e Heterozygote heterozygote one overlapping allele three peaks e Heterozygote heterozygote two overlapping alleles two peaks e 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 determine 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 the different allele peak
62. After Buffer and Enzyme Component Replacement Overview Experiments 98 As part of an ongoing program to exercise greater control over raw materials used in the AmpFSTR PCR Amplification Kits manufacturing of the AmpliTaq Gold enzyme 10X PCR Buffer II Tris KCl buffer components is transitioning from Roche Molecular Systems to Life Technologies Manufacturing of both components by Life Technologies will be conducted according to the same specifications used previously by Roche The in house components are established raw materials in our next generation kits for example the NGM NGM SElect and Identifiler Plus Kits We performed studies to compare the performance of the MiniFiler Kit containing the in house components updated kit with the performance of the original kit focussing on studies most relevant to forensic DNA testing see SWGDAM Guidelines effective January 1 2011 These studies while not exhaustive are in our opinion appropriate for a manufacturer Our studies compared the performance of two Roche manufactured enzyme and buffer lots Control mixes with three new lots of buffer and two new lots of enzyme manufactured by Life Technologies Test mixes Studies were performed using Test mixes containing both the enzyme and buffer manufactured by Life Technologies Control A mix Control mix Test A mix Test B mix Test C mix Material Buffer Control Buffer Control Buffer Test Buffer
63. CEPH family DNA sets are ideal for studying inheritance patterns Begovich et al 1992 Three CEPH family DNA sets were examined 0 50 ng of DNA from each sample was amplified using the MiniFiler Kit and Identifiler Kit followed by analysis using Applied Biosystems 3130x Genetic Analyzer The families examined included 1333 9 offspring 1340 7 offspring and 1345 7 offspring representing 23 meiotic divisions The results showed concordance between MiniFiler Kit and Identifiler Kit genotypes and confirmed that the loci are inherited according to Mendelian rules as expected The MiniFiler Kit loci have been mapped and the chromosomal locations have been published Nakahori et al 1991 Edwards et al 1992 Kimpton et al 1992 Mills et al 1992 Sharma and Litt 1992 Li et al 1993 Straub et al 1993 Barber and Parkin 1996 AmpFtSTR MiniFiler PCR Amplification Kit User Guide 81 g lt e 3 e 5 5 E s 2 Chapter 5 Experiments and Results Species specificity Species specificity SWGDAM Guideline 2 2 For techniques designed to type human the potential to detect from forensically relevant nonhuman species should be evaluated SWGDAM July 2003 The MiniFiler Kit provides the required specificity for primates Other species do not amplify for the loci tested Nonhuman studies The following species were tested in the specified
64. CR Amplification Kit User Guide 63 Chapter 5 Experiments and Results 64 AmpFtSTR MiniFiler PCR Amplification Kit User Guide Section 5 1 Overview Experiments using the MiniFiler Kit Importance of validation Experiment conditions Section 5 1 Developmental Validation Overview Developmental Validation This chapter provides results of the developmental validation experiments we performed using the AmpF STR MiniFiler PCR Amplification 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 that are critical for sound data interpretation in casework Sparkes Kimpton Watson et al 1996 Sparkes Kimpton Gilbard et al 1996 Wallin et al 1998 The experiments to evaluate the performance of the MiniFiler Kit were performed according to the DNA Advisory Board DAB Quality Assurance Standards effective October 1 1998 DNA Advisory Board 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 We performed additional experiments according to the revised guidelines from the Scientific Working Group on DNA Anal
65. Control mixes but did not exceed 50 RFU Figure 22 Reproducibility study known artifact VIC9 dye Y scale 50 RFU VIC9 dye labeled artifacts at 115 bp Control A zik ak dan n 7 Mark Sample For Deleti Control B m ak 7 Mark Sample for Deletion Test A J o aa aman dah a nan asi m Ac Mark Sample for Deletion TestB Mark Sample for Deleti Test A UU 100 AmpFtSTR MiniFiler PCR Amplification Kit User Guide Section 5 2 Performance Validation After Buffer Enzyme Component Replacement Sensitivity study Sensitivity study For the sensitivity study dilution series of three genomic DNA samples were amplified 0 75 ng three replicates 0 5 ng 0 25 ng and 0 125 four replicates each The results were evaluated for mean referenced peak height degree of linearity between input DNA concentration and peak height level of allelic dropout at 125 pg and genotype concordance Mean referenced Overall mean referenced peak height observations were consistent between all Test peak height and Control mixes Figure 23 demonstrating equivalent performance Figure 24 Figure 23 Sensitivity study mean peak heights for three genomic DNA samples 125 250 500 750 Lot Control Control 9 Test A Test B Te
66. Developmental Validation Stability Figure 17 Amplification of Control DNA 007 in the presence of hematin analyzed on the Applied Biosystems 3130xl Genetic Analyzer 1 ng DNA 007 Untreated lt Identifiler Kit 2 3 1 ng DNA 007 80 uM Hematin m lt a 2 2 ng DNA 007 Untreated SGM Plus Kit 2 ng DNA 007 80 uM Hematin 1 ng DNA 007 Untreated MiniFiler Kit 1 ng DNA 007 80 uM Hematin Comparison of performance of the three kits in a simulated model of hematin inhibition Only those loci gt 50 RFU represented in the MiniFiler Kit were measured in the Identifiler Kit and SGM Plus Kit see Table 6 A complete profile with Control DNA 007 yields 17 peaks using the MiniFiler Kit AmpFtSTR9 MiniFiler PCR Amplification Kit User Guide 87 Chapter 5 Experiments and Results Stability Effect of inhibitors Humic Acid 88 Table 6 Comparison of MiniFiler Identifiler and SGM Plus Kit performance in simulated model of hematin inhibition 3 Hematin MiniFiler Kit Identifiler Kit SGM Plus kit 20 uM 17 17 17 17 17 17 17 17 17 17 17 17 14 14 14 14 14 14 40 uM 17 17 17 17 17 17 17 17 17 17 9 17 14 14 14 14 14 14 60 uM 17 17 17 17 17 17 2 17 2 17 0 17 2 14 1 14 2 14 80 uM 17 17 17 17 17 17 0 17 0
67. E QE dO E SA 65 Developmental validation 65 SWGDAM guideline 1 2 1 2 2 i gt c sid kak nad W REGE 65 SWGDAM guideline 2 10 1 65 PGR COMPOMCMIS s n dixa bunte 66 Thermal cycler parameters 66 PGR cycle cite ce TC Tes necp Ie EN Re Lads 67 Accuracy precision and reproducibility 68 SWGDAM guideline 2 9 68 ee 68 Precision and size 5 69 Extra peaks in the electropherogram 75 Causesofextra peaks 25 22 td RARI RE RE RR HERE E RE E EE 75 Artifacts E 79 Characterizationiof loci 2 ee lak wa ika 81 SWGDAM guideline 2 1 2 81 Nature of the polymorphisms 81 Inheritance eri UR ARRIERE E e EUR 81 Mapping Renee Re EU Met bad ba deo RI TOS 81 Species Specificity i eei p RID headed Wee b eb tard e REC Ib T 82
68. L 102251338 D21511 70 90 10 40 4120 130 14 450 160 174 180 192 200 40 220 20 24 250 260 20 280 290 10165539 1018551 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 1600 800 0 CSFIPO FCA 70 80 30 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 _ _ _ ie 7 lel pss AmpFtSTR MiniFiler PCR Amplification Kit User Guide 13 1 Chapter 1 Overview Workflow overview Workflow overview Perform Bik T PCR 83 e xa es w e 5 AutoMate Express System PrepFiler Express Kit m Ea o gt E 8 a fey 1 Quantifiler Duo DNA Quantification Kit 55 9 9 AmpF STR MiniFiler PCR Amplification Kit ag Di E e Az te N GeneAmp PCR System 9700 Cycler Veriti 96 Well Thermal Cycler Perform u ha 3 3 phoresis 3100 3100 Avant 3130 3130xl 3500 3500xL
69. L Genetic Analyzer Quick Reference Card 4401662 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Data Collection v1 0 4401661 Life Technologies 3500 3500xL Genetic Analyzer User Bulletin Solutions to issues related to software data 4445098 hardware and consumables Note Additional user bulletins may be available at www lifetechnologies com Life Technologies 3730 3730xl Genetic Analyzer Getting Started Guide 4359476 GeneAmp PCR System 9700 Base Module User s Manual N805 0200 Quantifiler Kits Quantifiler Human DNA Quantification Kit and Quantifiler Y Human Male DNA 4344790 Quantification Kit Users Manual GeneMapper ID Software Version 3 1 Human Identification Analysis User Guide 4338775 GeneMapper ID Software Versions 3 1 and 3 2 Human Identification Analysis Tutorial 4335523 Installation Procedures and New Features for GeneMapper ID Software v3 2 User Bulletin 4352543 GeneMapper ID X Software Version 1 0 Getting Started Guide 4375574 GeneMapper ID X Software Version 1 0 Quick Reference Guide 4375670 GeneMapper ID X Software Version 1 0 Reference Guide 4375671 GeneMapper ID X Software Version 1 1 Mixture Analysis Getting Started Guide 4396773 GeneMapper ID X Software Version 1 1 Mixture Analysis Quick Reference Guide 4402094 GeneMapper ID X Software Version 1 2 Reference Guide 4426481 GeneMapper ID X Software Version 1 2 Quick Reference Guide 4426482 AmpFtSTR
70. Range All Sizes Y internal R validation G P studies to determine Smoothing and Baselining settings Min Peak Half Width Smoothing None Light Polynomial Degree Heavy Peak Window Size Baseline Window 51 Slope Threshold Peak Start 10 0 10 0 5 Calling Method 2nd Order Least Squares Normalization as xus Use Normalization if applicable Local Southern Met Global Southern Method Factory Defaults IMPORTANT Perform the appropriate internal validation studies to determine the appropriate peak amplitude thresholds for interpretation of MiniFiler Kit data Fields include Peak amplitude thresholds The software uses these parameters to specify the minimum peak height in order to limit the number of detected peaks Although GeneMapper ID X Software displays peaks that fall below the specified amplitude in electropherograms the software does not label or determine the genotype of these peaks Size calling method The MiniFiler Kit has been validated using the 3rd Order Least Squares sizing method with the 5 500 1179 Size Standard If you use the GeneScan 600 LIZ Size Standard v2 0 select the Local Southern Method Select alternative sizing methods only after performing the appropriate internal validation studies Normalization A Normalization checkbox is available on this tab i
71. S600v2 0Analysis User Bulletin Part 4332345 Applied 3 0 Windows e HlDFragmentAnalysis36 POP4 1 Applied Biosystems 3130 3130 Biosystems 9 XP Injection conditions Genetic Analyzers Using Data 3130 3130xlt 3130 3 kV 5 sec Collection Software v3 0 Protocols for Processing AmpFtSTR PCR 3130 3 kV 10 sec Amplification Kit PCR Products User Dye Set 65 Bulletin Part no 4363787 t We conducted validation studies for the MiniFiler Kit using the 3130xl configuration AmpFtSTR MiniFiler PCR Amplification Kit User Guide 27 o 9 ES P gt S 3 m 5 a 8 ER 5 0 ra E 5 2 Chapter 3 Perform Electrophoresis Prepare samples for electrophoresis on the 3100 3100 Avant or 3130 3130xl instruments Prepare samples for electrophoresis on the 3100 3100 Avant or 3130 3130xl instruments Prepare the samples for electrophoresis immediately before loading 1 0 pesce ON 28 Calculate the volume of Hi Di Formamide and size standard needed to prepare the samples Reagent volume per Reagent reaction reaction GeneScan 500 0 3 uL OR GeneScan 600 J O 5yuL LIZ9 Size Standard 1179 Size Standard v2 0 Hi Di Formamide 870 Hi Di Formamide 85 Note Include additional samples your calculations to volume for the
72. Test Buffer Test Buffer Lot 1 Lot 2 Lot 1 Lot 2 Lot 3 Enzyme Control Control TestEnzyme Control Test Enzyme Enzyme Enzyme Lot 1 Enzyme Lot 2 Lot 1 Lot 2 Lot 2 Each of the five mixes listed above were used to conduct reproducibility sensitivity degraded DNA and inhibition studies All amplifications were performed using a GeneAmp PCR System 9700 with either silver or gold plated silver block using the recommended amplification conditions and cycle number for the MiniFiler Kit All data was run on an Applied Biosystems 3130x Genetic Analyzer running Data Collection Software v3 0 and analyzed using GeneMapper ID X Software Subsequent data analysis was performed using Minitab Statistical Software AmpFtSTR MiniFiler PCR Amplification Kit User Guide Section 5 2 Performance Validation After Buffer Enzyme Component Replacement Reproducibility study Reproducibility study For the reproducibility study 12 replicates of control 007 at 0 5 ng input and three negative control replicates were amplified The results were evaluated for intracolor balance stutter percentage and the presence signal intensity and location of artifacts balance No significant difference lt 10 increase or decrease in the level of intracolor balance was observed between the Test and Control mixes with the exception of Control B Mix which showed slightly increased levels of intracolor balance for t
73. USER GUIDE applied biosystems by Life technologies AmpF STR MiniFiler PCR Amplification Kit kit Part no 4373872 Publication Part Number 4374618 Rev F Revision Date August 2012 2 technologies For Forensic Paternity Use Only Information in this document is subject to change without notice LIFE TECHNOLOGIES CORPORATION AND OR ITS AFFILIATE S DISCLAIM ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT EXPRESSED OR IMPLIED INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY FITNESS FOR A PARTICULAR PURPOSE OR NON INFRINGEMENT TO THE EXTENT ALLOWED BY LAW IN NO EVENT SHALL LIFE TECHNOLOGIES AND OR ITS AFFILIATE S 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 TRADEMARKS The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners Windows and Windows Vista are registered trademarks of Microsoft Corporation FTA is a registered trademark of Whatman International Ltd TaqMan is a registered trademark of Roche Molecular Systems Inc 2012 Life Technologies Corporation All rights reserved Contents About This Guide c ks sk kk kk kk kk kk kk KK KK KK KK kk kk kk kok kk kok kk kk kk 9 Wo to AAAA ite dtm 9 Purpose HHHH HHHH EXTA EN te
74. Validation After Buffer and Enzyme Component Replacement Inhibition study Figure 34 Inhibition study intracolor balance Lot Control Control B Test A A Test B p Test 2 9 Li 9 o o x E Panel variable Inhibitor Representative electropherograms from the inhibition study are shown in Figure 35 Figure 36 and Figure 37 Figure 35 Inhibition study representative electropherograms using uninhibited Control DNA 007 Y scale 3000 RFU 75 95 115 135 155 175 195 215 235 255 3000 Control A Mark Sample for Deletion 75 35 115 135 155 175 195 215 235 255 3000 2000 Control 10004 0 Mark Sample for Deletion 75 35 115 135 155 175 195 215 235 255 3000 Mark Sample for Deletion 75 95 115 135 155 175 195 215 235 255 3000 Tl Test Mark for Deletion 75 95 115 135 155 175 195 215 235 255 3000 Test 10007 0 110 AmpFtSTR MiniFiler PCR Amplification Kit User Guide Section 5 2 Performance Validation After Buffer Enzyme Component Replacement Inhibition study Figure 36 Inhibition study representative electropherograms using Control DNA 007 inhibited with 45 uM Hematin Y scale 3000 RFU 75 3000
75. allest standard deviations in sizing Figure 7 on page 69 illustrates the tight clustering of allele sizes obtained on the AmpFtSTR MiniFiler PCR Amplification Kit User Guide 69 70 Chapter 5 Experiments and Results Accuracy precision and reproducibility 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 in the final allele assignment GeneMapper ID Software and GeneMapper ID X Software automatically flag sample alleles that do not size within the prescribed window around an allelic ladder allele by labelling the allele as OL off ladder Maximum sizing precision is obtained within the same set of capillary injections Cross platform sizing differences occur due to a number of factors including type and concentration of polymer run temperature and electrophoresis conditions Variations in sizing can also occur between runs on the same instrument and between runs on
76. ation Kit User Guide 85 Chapter 5 Experiments and Results Stability Effect of inhibitors Hematin 86 Table 5 Comparison of MiniFiler Identifiler and SGM Plus Kit performance in simulated model of DNA degradation 3 DNase MiniFiler Kit Identifiler Kit SGM Plus kit 0 units 14 14 14 14 14 14 14 14 14 14 14 14 10 10 10 10 10 10 4 units 14 14 14 14 14 14 8 14 3 14 4 14 2 10 4 10 5 10 5 units 14 14 14 14 14 14 3 14 4 14 4 14 2 10 2 10 2 10 6 units 14 14 14 14 13 14 0 14 0 14 0 14 0 10 1 10 1 10 Heme compounds have been identified as PCR inhibitors in DNA samples extracted from bloodstains DeFranchis et al 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 MiniFiler Kit male Control DNA 007 1 ng input DNA for the MiniFiler Kit and the Identifiler Kit and 2 ng for the SGM Plus Kit was amplified with increasing concentrations of hematin The concentrations of hematin used were 0 uM 20 uM 40 uM 60 uM and 80 uM No preferential amplification was observed in the presence of increasing amounts of hematin Figure 17 on page 87 AmpFtSTR MiniFiler PCR Amplification Kit User Guide Section 5 1
77. avelength there is some overlap in the emission spectra between the dyes Figure 2 The goal of multicomponent analysis is to correct for spectral overlap AmpFtSTR MiniFiler PCR Amplification Kit User Guide 15 1 Chapter 1 Overview Materials and equipment Figure 2 Emission spectra of the five dyes used in the MiniFiler Kit Dyes 6 FAM VIC NED PET LIZ A B O O O N o 2 2 o N T 2 9 2 o Wavelength nm Materials and equipment Kit contents and The MiniFiler Kit Part no 4373872 contains sufficient quantities of the following storage reagents to perform 100 reactions at 25 uL reaction IMPORTANT The fluorescent dyes attached to the primers are light sensitive Protect the primer set amplified DNA allelic ladder and size standard from light when not in use Keep freeze thaw cycles to a minimum Table 2 Kit contents and storage Component Description 100 reaction Storage AmpF STR9 MiniFiler Contains enzyme salts dNTPs carrier 2 tubes 15 to 25 C upon Master Mix protein and 0 05 sodium azide 0 5 mL tube receipt 2 to 8 C after AmpF STR Control Contains 0 10 ng uL human male genomic 1 tube 0 3 mL 007 DNA 0 05 sodium azide and buffer t See Table 1 on page 12 for profile AmpF STR MiniFiler Contains forward and reverse primers to 1 tube 0 5 mL 15 to 25 C upon Primer Set amplify human DNA targets
78. complete A nucleotide addition These data were generated on the 310 Genetic Analyzer using another AmpF STR kit Bav 175 180 185 190 195 900 Cun No Exter sion 300 200 700 2 600 5 500 3 A 400 D E 5 100 Final Extension Lack of complete A nucleotide addition may be observed in MiniFiler Kit results when the amount of input DNA is greater than the recommended protocols because more time is needed for the DNA Polymerase to add the A nucleotide to all molecules as more PCR product is generated Amplification of too much input DNA may also result in off scale data Artifacts Artifacts and anomalies are seen in all molecular biological systems Artifacts are typically reproducible while anomalies are non reproducible intermittent occurrences that are not observed consistently in a system for example spikes and baseline noise Artifacts have been seen in data produced on genetic analyzers when using the MiniFiler Kit In amplified samples artifacts in the non calling region may appear in the blue 70 nt and VIC 80 nt dyes Low level artifacts in the calling region may appear in the blue 117 and 127 nt green 118 nt and black 166 nt dyes depending on the sensitivity of the instrument AmpFtSTR9 MiniFiler PCR Amplification Kit User Guide 79 80 Chapter 5 Experiments and Results Extra peaks in the electropherogram
79. complete list of parts and accessories for the 3500 3500xL instrument refer to the Applied Biosystems 3500 3500xL Genetic Analyzer User Guide PN 4401661 118 AmpFtSTR MiniFiler PCR Amplification Kit User Guide Appendix Ordering Information Equipment and materials not included Source 310 Analyzer materials 310 DNA Analyzer capillary array 47 cm 402839 0 5 mL sample tray 5572 96 well tray adaptor for 9700 thermal cycler trays 4305051 GeneScan 500 LIZ Size Standard 4322682 OR OR GeneScan 600 LIZ Size Standard v2 0 4408399 Running Buffer 10X 4335643 Genetic analyzer septa retainer clips for 96 tube sample tray 402866 Genetic analysis sample tubes 0 5 mL 401957 Septa for 0 5 mL sample tubes 401956 05 33 Matrix Standard Set 6 FAM VIC PET and 1129 dyes for 310 377 systems 4318159 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 For a complete list of parts and accessories for the 310 instrument refer to Appendix B of the 370 Genetic Analyzer User Guide Pub no 4317588 PCR Amplification MicroAmp 96 Well Tray N8010541 MicroAmp Reaction Tube with Cap 0 2 mL N8010540 MicroAm
80. d Results Accuracy precision and reproducibility Allele Mean Standard Deviation 13 177 48 177 50 0 041 0 047 14 181 46 181 49 0 034 0 050 15 185 45 185 47 0 034 0 053 17 150 52 150 55 0 031 0 040 18 154 26 154 29 0 031 0 043 19 158 03 158 04 0 029 0 047 20 161 78 161 80 0 033 0 044 21 165 55 165 57 0 030 0 042 22 169 32 169 34 0 031 0 047 23 173 11 173 12 0 032 0 041 24 176 88 176 91 0 034 0 048 25 180 68 180 70 0 025 0 045 26 184 49 184 51 0 031 0 047 26 2 186 29 186 34 0 027 0 049 27 188 34 188 37 0 022 0 047 28 192 20 192 25 0 037 0 047 29 195 97 196 02 0 032 0 046 30 199 69 199 74 0 032 0 047 30 2 202 12 202 17 0 034 0 055 31 2 205 94 205 98 0 034 0 055 32 2 209 74 209 80 0 034 0 051 33 2 213 57 213 64 0 035 0 064 42 2 248 46 248 55 0 042 0 064 43 2 252 35 252 43 0 038 0 067 44 2 256 39 256 46 0 043 0 064 45 2 260 28 260 36 0 043 0 054 46 2 263 89 263 95 0 040 0 055 47 2 267 71 267 77 0 039 0 057 48 2 271 69 271 76 0 040 0 058 50 2 279 48 279 54 0 036 0 062 51 2 283 23 283 28 0 041 0 061 74 AmpFtSTR MiniFiler PCR Amplification Kit User Guide Section 5 1 Developmental Validation Extra peaks in the electropherogram Extra peaks in the electropherogram Causes of extra peaks Peaks other than the target alleles may be detected on the electropherogram Causes for the appearance of extra peaks include
81. d by the frequency of that genotype in the relevant population s The MiniFiler Kit contains loci for which extensive population data are available For additional information on 11 loci shared between many of the AmpF STR kits see the population data and additional studies section of the AmpF STR Identifiler PCR Amplification Kit User Guide Part no 4323291 Analyzing the four databases Analysis across the four databases of 2274 total chromosomes per locus revealed the following number of different alleles 10 CSF1PO alleles 13 0251338 alleles 9 075820 alleles 8 0135317 alleles 8 0165539 alleles 20 018551 alleles 26 021511 alleles and 31 FGA alleles AmpFtSTR MiniFiler PCR Amplification Kit User Guide 93 g lt o e 3 e 5 2 s Chapter 5 Experiments and Results Population data Concordance studies 94 In addition to the alleles that were observed and recorded in the Life Technologies databases other alleles have been published or reported to us by other laboratories see the STRBase at www cstl nist gov div831 strbase Low frequency alleles Some alleles of the MiniFiler Kit loci occur at a low frequency For these alleles minimum frequency five divided by 2n where n equals the number of individuals in the database was assigned for the MiniFiler Kit African American U S Caucasian U S Hispanic and Native American databases as suggested in the 1996 repo
82. dit Size Standard Description Name MiniFiler_GS500 Security Group GeneMapper ID X Security Group Ekg Description Size Standard Dye r Size Standard Table Size in Basepairs di GERE 2 AmpFtSTR MiniFiler PCR Amplification Kit User Guide 59 Chapter 4 GeneMapper ID X Software Analyze and edit sample files with GeneMapper ID X Software Analyze and edit sample files with GeneMapper D X Software 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 Settings Sample Type Select the sample type Analysis Method MiniFiler AnalysisMethod v2X the name of the analysis method you created Panel AmpFLSTR Panels v2X Size Standard CE_G5_HID_GS500 or the name of the size standard you created Note For more information about how the Size Caller works refer to the TM GeneScan Analysis Software for the Windows9 NT Operating System Overview of the Analysis Parameters and Size Caller User Bulletin Pub no 4335617 60 AmpFtSTR MiniFiler PCR Amplification Kit User Guide Section 4 2 Software Examine and edit a project 3 Click Analyze enter a name for the project in the Save Project dia
83. dow of any known allelic ladder allele or virtual bin Note If a sample allele peak is called as an off ladder allele the sample result needs to be verified according to the laboratory s protocol AmpFtSTR MiniFiler PCR Amplification Kit User Guide Section 4 2 Software 4 Set up GeneMapper ID X Software for data analysis Set up GeneMapper D X Software for data analysis Panel bin and The file names shown in this section may differ from the file names you see when you stutter file version download or import files If you need help determining the correct files to use contact your local Life Technologies Human Identification representative or go to www lifetechnologies com support Software Patches amp Updates GeneMapper ID X Software The instructions and examples in this section refer to the latest version of panel bin and stutter file available at the time of publication Before using the Before you use GeneMapper ID X Software v1 0 1 or higher for fsa files v1 2 or software for the higher for hid files to analyze data for the first time you must do the following first time 1 Check the version of panel bin and stutter files installed with the GeneMapper ID X Software as explained in Check panel bin and stutter file version below C 0 2 0 lt i 0 e v gt e p 2 0 2 Check www lifetechnologies com support gt
84. dows were tested to verify that a specific PCR product with the desired sensitivity of at least 0 50 ng of Control DNA 007 was produced For example annealing temperatures of 55 57 59 61 and 63 C were tested for two minute hold times the Silver 96 Well GeneAmp PCR System 9700 Figure 5 The PCR products were analyzed using an Applied Biosystems 3130xl Genetic Analyzer 66 AmpFtSTR MiniFiler PCR Amplification Kit User Guide Section 5 1 Developmental Validation Developmental validation Of the tested annealing temperatures 55 to 61 C produced robust profiles At 63 C the yield of the majority loci was significantly reduced No preferential amplification was observed at the standard annealing temperature of 59 C Thermal cycler temperature is critical to assay performance therefore routine regularly scheduled thermal cycler calibration is strongly recommended Figure 5 Electropherograms obtained from amplification of 0 50 ng of Control DNA 007 at annealing temperatures of 55 57 59 61 and 63 C analyzed on an Applied Biosystems 3130xl Genetic Analyzer y axis 4000 RFU 70 80 30 100 110 120 130 140 150 160 170 180 130 200 210 220 230 240 250 260 270 280 290 70 80 80 100 110 120 130 140 150 160 170 180 130 200 210 220 230 240 250 260 270 280 290 g lt e 3 o e 5 E 2 70 80 30 100 110 120 130 140 150 160 170 180 130 200 210 220 230
85. e AmpFLSTR Panels v2X folder to display its list of kits in the right pane b Double click the MiniFiler v1 1X folder to display its list of markers below it c Double click 021511 to display the Stutter Ratio amp Distance view for the marker in the right pane Panel Manager File Edit Bins View Help mx HN B inset AmpFLSTR Bins 2 WI HuL ESE o m Cz Identifiler CODIS v1 1X DEAE R a Cp Identifiler Direct_v1 1X Please enter the stutter filter s for D21511 marker here If left blank the global stutter filter m C Identifiler Plus v1 1x will be applied m Identifiler_v1 1X MiniFiler_v1 1X Minus Stutter Plus Stutter Gt 075820 From Distance To Distance E AMEL T 3 25 4 75 2 021511 From Distance To Distance Stutter Ratio amp Distance 0165539 018551 lt CSF1PO E New Edit Delete af Reference Samples 11 Click Apply then OK to add the MiniFiler Kit panel bin set and marker stutter to the GeneMapper ID X Software database IMPORTANT If you close the Panel Manager without clicking Apply the panels bin sets and marker stutter will not be imported into the GeneMapper ID X Software database 52 AmpFtSTRO MiniFiler PCR Amplification Kit User Guide Section 4 2 Software 4 Set up GeneMapper ID X Software for data analysis Crea
86. e silver or gold plated silver 96 well blocks or a Veriti 96 Well Thermal Cycler AmpFtSTR MiniFiler PCR Amplification Kit User Guide 21 Perform Perform PCR Chapter 2 Perform PCR 1 Program the thermal cycling conditions When using the GeneAmp PCR System 9700 with either 96 well silver or gold plated silver block select the 9600 Emulation Mode When using the Veriti 96 Well Thermal Cycler refer to the following document for instructions on how to configure the Veriti instrument to run in the 9600 Emulation Mode User Bulletin Veriti 96 Well Thermal Cycler AmpFtSTRO Kit Validation Pub no 4440754 Initial Denature Anneal Extend Final Final hold incubation step extension HOLD CYCLE 30 HOLD HOLD 95 94 59 72 60 4 C 11 min 20 sec 2 min 1 min 45 min 2 Load the plate into the thermal cycler and close the heated cover IMPORTANT If using the 9700 thermal cycler with silver or gold plated silver block and adhesive clear film instead of caps to seal the plate wells be sure to place MicroAmp compression pad Part no 4312639 on top of the plate to prevent evaporation during thermal cycling The Veriti Thermal Cycler does not require a compression pad 3 Start the run 4 On completion of the run store the amplified DNA and protect from light If you are storing the DNA Then place at 2 to 8 C 15 to 25 C lt
87. ea 121 Amplified DNA work area 122 APPENDIX E Safety 123 Chemical Safty De doe coh a t b 124 Specific chemical handling lt ken kun k 124 Biological hazard 125 Bibli gra phy e EDD dare ames ware Sears 127 Documentation and 131 Related documentation 4 2 2 131 Obtain S DS 5 2 ie hes MM Han Pad pa Sea eho 132 Obtain support ose d pot HnHNHHEHN NE EE es 132 Limited Product Warranty WW kk kk kk kk kk kk kK kk kk KK kK KK KK kk kk kK kk kk kk kk kk kk kk kk kK kk kk 132 lap od acme lee nae DA Waa ede b ae 133 AmpFtSTR MiniFiler PCR Amplification Kit User Guide 7 Contents 8 AmpFtSTR MiniFiler PCR Amplification Kit User Guide About This Guide IMPORTANT Before using this product read and understand the information the Safety in this document Revision history Revision Date Description A October 2006 Ne
88. ea ete ee E EEE ee ee el 9 B CHAPTER 1 11 Prod ctoverview 5207 REN ute cite 11 Bil g olo SORAN eee etree I MNA Md E cad eae ge 11 5 ates deat ter et nist E Ah 11 About the primers zs iti EE led di NEJME ID 11 Loci amplified by the kit 12 Allelic ladder idee eda dede ete e P Re 13 WorktloW OVerVIe W oU tI deni LA et Bug estan s iae 14 Instrument and software overview 15 Data collection and analysis software 15 Instrument and software compatibility 15 About multicomponent analysis 15 How multicomponent analysis works 15 Materials and equipment 16 Kit contentsvand storage wa kl kay A ERA ia eei 16 Standards for samples 17 B CHAPTER 2 Perform 19 Required user supplied reagents
89. efaults In the Bin Set field select the AmpFLSTR_MiniFiler_Bins_v1 bin set imported previously and configure the stutter distance parameters as shown GeneMapper ID Software v3 2 1 allows you to specify four types of marker repeat motifs tri tetra penta and hexa You can enter parameter values for each type of repeat in the appropriate column Specify the peak filter ratios To apply the stutter ratios listed in the Allele tab for single source data deselect the Use marker specific stutter ratio if available check box selected by default Perform appropriate internal validation studies to determine the appropriate filter setting to use Note Applying global stutter ratios may reduce the editing required for single source sample data To apply the stutter ratios contained in the MiniFiler GS500 v1 file select the Use marker specific stutter ratio if available check box selected by default Perform appropriate internal validation studies to determine the appropriate filter setting to use To specify an Amelogenin Cutoff Ratio enter the appropriate ratio into the Amelogenin Cutoff field Note Do not use an Amelogenin cutoff for data that may contain mixtures of male and female DNA AmpFtSTR9 MiniFiler PCR Amplification Kit User Guide 39 4 Chapter 4 Analyze Data Set up GeneMapper ID Software for data analysis Peak Detector tab settings Analysis Method Editor HID General A
90. ell Sample Block N8050251 Gold plated Silver 96 Well Sample Block 4314443 Tabletop centrifuge with 96 Well Plate Adapters optional MLS major laboratory supplier Table 16 User supplied materials Source AmpFfSTR MiniFiler PCR Amplification Kit 4373872 3100 Analyzer materials 96 Well Plate Septa 4315933 Reservoir Septa 4315932 3100 3130xl Genetic Analyzer Capillary Array 36 cm 4315931 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 OR OR GeneScan 600 LIZ Size Standard v2 0 4408399 Running Buffer 10X 402824 Hi Di Formamide 4311320 DS 33 Matrix Standard Kit Dye Set G5 4345833 MicroAmp Optical 96 Well Reaction Plate N8010560 250 uL Glass Syringe array fill syringe 4304470 AmpFtSTR MiniFiler PCR Amplification Kit User Guide 117 Appendix C Ordering Information Equipment and materials not included Source 5 0 mL Glass Syringe polymer reserve syringe 628 3731 For a complete list of parts and accessories for the 3100 instrument refer to Appendix B of the 3700 Genetic Analyzer and 3100 Avant Genetic Analyzer User Reference Guide Part no 4335393 3130xl Analyzer materials 96 Well Plate Septa 4315933 Reservoir Septa 4315932 3100 3130xl Genetic Analyzer Capillary Array 36 cm 4315931 POP 49 Polymer for 3130 313
91. ence 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 calculated percent stutter g lt e 3 D e 5 E e 2 Multicomponent analysis of off scale data is not accurate This inaccuracy results in poor spectral separation pull up Incomplete A nucleotide addition The sample can be re amplified 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 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 Figure 15 Effect of amplifying varying amounts of Control DNA 007 70 80 30 100 410 4204180 44 459 40 10 40 1 200 40 20 230 240 250 260 270 2790 290 0 A M M 4000 1n g 2000 0 2 2 2 22 2 2 2 2 2 70 4000 0 50 ng 0 25 ng 120 0 125 ng 0 0 062 ng Note that the y axis scale is magnified for the lower amounts of DNA analyzed using the Applied Biosystems
92. ensic Laboratories Handbook for Facility Planning Design Construction and Moving National Institute of Justice 1998 Parentage DNA testing refer to the Guidance for Standards for Parentage Relationship Testing Laboratories American Association of Blood Banks 7th edition 2004 The sensitivity of AmpF STR Kits and other PCR based tests enables amplification of minute quantities of DNA necessitating precautions to avoid contamination of samples yet to be amplified Kwok and Higuchi 1989 Also take care while handling and processing samples to prevent contamination by human DNA Wear gloves at all times and change them frequently Close sample tubes when not in use Limit aerosol dispersal by handling sample tubes and reagents carefully Note We do not intend these references for laboratory design to constitute all precautions and care necessary for using PCR technology PCR setup work area IMPORTANT These items should never leave the PCR Setup 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 e Pipette tips sterile disposable hydrophobic filter plugged Pipettors AmpFtSTR MiniFiler PCR Amplification Kit User Guide 121 Appendix D PCR Work Areas Amplified DNA work area Tube decapper autoclavable e Vortex Amplified DNA w
93. er 4 Find then open the folder containing the panels bins and marker stutter a Select Panel Manager in the navigation pane 22 Panel Manager b Select File Import Panels to open the Import Panels dialog box File Edit Bins View 5 rei c Navigate to then open the AmpFLSTR Analysis i a mi Files GMIDX folder that you unzipped in step 1 B Panel Manager on page 48 48 AmpFtSTR MiniFiler PCR Amplification Kit User Guide Section 4 2 Software 4 Set up GeneMapper ID X Software for data analysis 5 Select AmpFLSTR_Panels_v2X or the version you installed then click Import Note Importing this file creates a new folder in the navigation pane of the Panel Manager AmpFLSTR_Panels_v2X This folder contains panels for multiple 5 kits and associated markers Import Panels Lookin G AmpFLSTR Analysis Files GMIDX cones B AmpFLSTR_Bins_v2X My Recent Ej ampFLSTR stutter vzx Documents Dl ReadMe AmpFLSTR 2 My Documents ys File name AmpFLSTR_Panels_v2X txt My Computer Files of type All Files C 0 2 lt D 0 e v e p 2 0 6 Import AmpFLSTR Bins v2X txt a Select the AmpFLSTR Panels v2X folder in the navigation pane Panel Manager File Edit Bins Yiew Help i x E N Bin Set v W Se
94. er ID X Software for data analysis General tab settings Analysis Method Editor General allele Peak Detector Peak Quality SQ amp GQ Settings MiniFiler AnalysisMethod v2 In the Name field either type the name as shown or enter a name of your choosing In the Security Group field select the Security Group appropriate to your software configuration from the dropdown list The Description and Instrument fields are optional 54 AmpFtSTR MiniFiler PCR Amplification Kit User Guide Section 4 2 Software 4 Set up GeneMapper ID X Software for data analysis Allele tab settings Analysis Method Editor General Allele Peak Detector Peak Quality 50 amp GQ Settings Bin Set AmpFLSTR_Bins Use marker specific stutter ratio and distance if available Marker Repeat Type Tri Tetra Penta Global Cut off Value 0 0 0 0 0 0 Ratio 0 0 0 0 0 0 MinusA Distance 0 0 0 0 0 0 0 0 0 0 0 0 Global Minus Stutter Ratio 0 0 0 0 0 0 Global Minus Stutter Distance From 0 0 3 25 0 0 To 0 0 4 75 0 0 C 0 2 0 lt D 0 e v gt 122 e e 2 0 Global Plus Stutter Ratio 0 0 0 0 0 0 Global Plus Stutter Distance From 0 0 0 0 0 0 To 00 0 0 0 0 Amelogenin Cutoff 0 0 Range Filter Factory Defaults
95. ers are light sensitive Protect the primer set amplified DNA allelic ladder and size standard from light when not in use Keep freeze thaw cycles to a minimum wo cx e el En EX zi 0 3 7 Electrophoresis The following table lists Data Collection Software and the run modules that can be software setup and used to analyze Identifiler Kit PCR products For details on the procedures refer to the documents listed in the table reference documents Data Operatin Collection 9 Run modules conditions References System Software 3 11 Windows XP GS STR POP4 1mL G5 v2 md5 310 Genetic Analyzer User s Manual Windows Injection condition Pub no 4317588 3 0 Windows 15 kV 5 sec 310 Protocols for Processing AmpF STRO PCR NT and Amplification Kit Products with Microsoft Windows Windows NT Operating System User Bulletin 2000 Pub no 4341742 We conducted concordance studies for the Identifiler Kit using this configuration Prepare samples for electrophoresis on the 310 instrument Prepare the samples for electrophoresis immediately before loading 1 Calculate the volume of Hi Di Formamide and size standard needed to prepare the samples Reagent Volume per reaction GeneScan 500 1129 Size Standard or 0 5 uL GeneScan 600 1179 Size Standard v2 0 Hi Di Formamide 24 5 uL Note Include additional samples in your calculations to provide excess volu
96. es IMPORTANT Thawing is required only during first use of the Primer Set and PCR Reaction Mix After first use these reagents are stored at 2 to 8 C and do not require subsequent thawing Do not refreeze these reagents 3 Prepare the reaction mix Pipette the required volumes of components into an appropriately sized polypropylene tube 4 Vortex the reaction mix for 3 seconds then centrifuge briefly 5 Dispense 15 uL of the reaction mix into each reaction well of a MicroAmp Optical 96 Well Reaction Plate or each MicroAmp tube 6 Prepare the DNA samples DNA sample To prepare Negative control Add 10 uL of low TE buffer 10mM Tris 0 1mM EDTA pH 8 01 Test sample Dilute a portion of the test DNA sample with low TE buffer so that 0 5 to 0 75 ng of total DNA is in a final volume of 10 uL Add 10 uL of the diluted sample to the reaction mix Positive control Combine 5 uL of control DNA 0 1 ng uL with 5 uL of low TE buffer for a total volume of 10 uL The final sample concentration is 0 05 ng L Add to the reaction mix The final reaction volume sample or control plus reaction mix is 25 uL 7 Sealthe plate with MicroAmp Clear Adhesive Film or MicroAmp Optical Adhesive Film or cap the tubes 8 Centrifuge the tubes at 3000 rpm for about 20 seconds in a tabletop centrifuge with plate holders if using 96 well plates 9 Amplify the samples a GeneAmp PCR System 9700 with th
97. et AmpFLSTR MiniFiler 55500 5 amp iPanel Manager JAmpFLSTR_Ytiler_Panel_v2 ES AmpFLSTR MiniFiler GS500 Panels v1 p MiniFiler 0135317 075820 AMEL 0251338 021511 0165539 018551 CSF1PO FGA Maker Name 0135317 075820 Dye Color NE uM Control Alleles Marker E Marker Comments Ladder Alleles blue 11 014 none 8 9 10 11 12 13 14 blue 7 12 011 none 6 7 8 9 10 11 12 1 AMEL green 00 none XY 0251338 green 20 23 018 none 15 16 17 18 19 20 021511 green 28 31 016 none 24 24 2 252627 7 0165539 yellow 9 10 0 15 none 5 8 9 10 11 12 13 018551 yellow 12 15 048 none 7 9 10 10 2 11 12 CSF1PO red 1112 044 none 5 7 8 3 10 11 12 1 Reference Samples 36 red 24 26 015 none 17 18 13 20 21 22 gt AmpFtSTR MiniFiler PCR Amplification Kit User Guide Section 4 1 ID Software n Set up GeneMapper ID Software for data analysis 8 Select 0281338 to display the Bin view for the marker in the right pane Panel Manager File Edit Bins View m m Bin Set AmpFLSTR_M niFiler_cSsno_ v D ES Bi EM a m m m m m al 5 amp iPanel Manager 8 T AmpFLSTR Yfiler Panel v2 E f A AmpFLSTR MiniFiler GS500 Panels v1 E f2MiniFiler 55500 v1 0135317 075820
98. fer Enzyme Component Replacement Sensitivity study Figure 25 Sensitivity study linear regression plot of combined mean peak height for three genomic DNA samples iun Lot Contro 3 0 4 Control B 2 Test 2 Test B 2 5 M TeC 2 0 1 5 Mean Referenced Peak Height U D ED 3 3 o 2 e 0 5 a 2 00 e ra 2 oH m N lt 3 9 2 9 Em 5 e D 3 D 5 0 0 100 200 300 400 500 600 700 800 Concentration pg Allelic dropout Allelic dropout was only visible at 125 pg input DNA concentration Levels of allelic dropout at 125 pg were comparable across all Test and Control mixes and are compared in Table 12 Examples of allelic dropout are shown in Figure 26 and Figure 27 Table 12 Sensitivity study summary of allelic dropout observed at 125 pg input concentration Number of Number of Number of Percent of Reagent Mix sampies alleles alleles alleles dropped dropped Test A 3 51 2 4 Test B 4 68 4 6 Test 4 68 2 3 Control A 4 68 2 3 Control B 4 68 4 6 AmpFtSTR MiniFiler PCR Amplification Kit User Guide 103 5 Chapter 5 Performance Validation After Buffer and Enzyme Component Replacement Sensitivity study Figure 26 Sensitivity study electropherogram of 125 ng Sample 3 amplified with Control Mix Two alleles are below the analysis threshold of 5
99. g the SGM Plus PCR Amplification Kit As the DNA became increasingly degraded the larger size Identifiler Kit and SGM Plus Kit loci became undetectable However the amplification with the MiniFiler Kit resulted in an increased overall typing success rate AmpFtSTR MiniFiler PCR Amplification Kit User Guide Section 5 1 Developmental Validation Stability Figure 16 Amplification of Raji samples untreated or sonicated for 5 minutes and incubated with DNase 1 1 ng DNA Untreated Ludi dii aul bal Identifiler Kit NNNM lt am 1 ng DNA 3 e o 5 Units 3 DNase 1 9 5 dins fot m E e 2 ng DNA Untreated SGM Plus Kit auo 199 019039 59 59 150 190 195 200 20 220 29 20 29 280 29 280 290 30 30 so 2 ng DNA P 5 Units DNase s 1 ng DNA Untreated MiniFiler Kit T 1 ng DNA m 5 Units DNase The performance of the three kits was compared a simulated model of DNA degradation sonication and DNase I treatments Only those loci gt 50 RFUs represented in the MiniFiler Kit were measured in the Identifiler Kit and SGM Plus Kit see Table 5 A complete profile with Raji DNA yields 14 peaks using the MiniFiler Kit AmpFtSTR9 MiniFiler PCR Amplific
100. gf Panel Manager Panel Name Comment AmpFLSTR_NGMSElect_v2 Er v ha 2 AmpFLSTR_NGM_v3X SGM Plus v1 1X null i as panel v15 NGM_SElect_v2 1 null cofiler_v1 1 Identifiler Plus v1 1X null 2356 Plus 1 1 NGM v3 1X null Elect v2 18 Identifiler Direct v1 1X null Ee Profiler Plus v1 1X null amp C3Identifller Direct v1 1X Profiler v1 1X null amp Cj Profiler Plus v1 1x SEfiler Plus v1 1X null E Identifiller v1 1x null n MiniFiler_vi 1 null c amp C MiniFiler v1 1X Profiler Plus CODIS v1 1 null Profiler Plus CODTS 1 1 Vfiler v1 1X null AmpFtSTR MiniFiler PCR Amplification Kit User Guide Section 4 2 Software 4 Set up GeneMapper ID X Software for data analysis 8 Select and expand MiniFiler_v1 1X in the navigation pane then select 0135317 to display the Bin view for the marker in the right pane Panel Manager File Edit Bins View Help ae x j ampFisTR_Bins_v2x H Identifiler_CODIS_v1 1X H E Identifiler Direct v1 1X amp C Identifiler Plus v1 1X amp C Identifiler v1 1X amp C3MiniFiler v1 1X amp 075820 GH AMEL i 0251338 89021511 j 0165539 i 018551 i CSF1PO C gt lt o 19 o 2 0 gt lt 122 E af Reference Samples 85 87 89 91 93 95 37 99 101 103 105 107 109 111 113 115 117 119 121 123 125 127 129 131 133
101. he FAM dye blue but decreased intracolor balance results for the PET dye red Figure 20 The levels of intracolor balance obtained for all Test and Control mixes fall within the expected range of performance for the MiniFiler Kit Figure 20 Reproducibility study intracolor balance 1 0 s Control U EM 3 3 D 5 o 5 2 e 5 00 E D 5 a m N lt 3 2 Control D Q 3 2 2 Intracolor Percentage e Dye Color Stutter Stutter percentage results for each marker were comparable across all Test and Control percentages mixes Figure 21 AmpFtSTR MiniFiler PCR Amplification Kit User Guide 99 Chapter 5 Performance Validation After Buffer and Enzyme Component Replacement Reproducibility study Figure 21 Reproducibility study mean stutter percentage Mean of Stutter Percentage Amp Con Control Control B Dye Color and Locus Artifacts Known artifacts observed showed the same morphology signal intensity and location in all Test and Control mixes and do not exceed 50 RFU Figure 22 No new artifacts were observed in the Test mixes No artifacts were observed in the Test and Control mixes for the 6 FAM dye blue NED dye yellow and PET red dye A very low level artifact was visible in the VIC green dye at 115 bp for all Test and
102. iFiler PCR Amplification Kit User Guide Section 5 1 Developmental Validation Accuracy precision and reproducibility Allele Mean Standard Deviation 30 210 59 210 63 0 053 0 075 30 2 212 53 212 59 0 058 0 067 31 214 54 214 59 0 054 0 069 31 2 216 51 216 55 0 054 0 075 32 218 48 218 55 0 051 0 067 32 2 220 48 220 53 0 057 0 073 3 33 222 46 222 51 0 055 0 073 8 33 2 224 52 224 57 0 052 0 078 3 34 226 35 226 39 0 054 0 063 5 34 2 228 42 228 47 0 049 0 072 8 35 230 35 230 40 0 047 0 081 5 35 2 232 38 232 45 0 055 0 083 36 234 42 234 48 0 053 0 080 37 238 31 238 36 0 057 0 074 38 242 41 242 47 0 063 0 077 0251338 15 120 06 120 10 0 032 0 042 16 124 11 124 16 0 028 0 044 17 128 07 128 12 0 031 0 044 18 132 04 132 08 0 036 0 051 19 136 05 136 08 0 028 0 046 20 140 00 140 04 0 028 0 045 21 144 01 144 04 0 034 0 043 22 147 99 148 02 0 029 0 039 23 151 96 151 99 0 034 0 044 24 155 95 155 97 0 029 0 042 25 159 93 159 94 0 038 0 049 26 163 91 163 94 0 032 0 055 27 167 99 168 01 0 033 0 052 28 172 24 172 26 0 038 0 052 D7S820 6 149 69 149 73 0 032 0 051 7 153 65 153 68 0 036 0 051 8 157 62 157 65 0 031 0 051 9 161 59 161 62 0 032 0 057 10 165 55 165 57 0 035 0 046 11 169 53 169 54 0 037 0 050 12 173 50 173 52 0 034 0 055 AmpFtSTR MiniFiler PCR Amplification Kit User Guide 73 Chapter 5 Experiments an
103. ial STR typing kit J Forensic Sci 49 859 860 Edwards A Civitello Hammond and Caskey 1991 DNA typing and genetic mapping with trimeric and tetrameric tandem repeats Am J Hum Genet 49 746 756 Edwards A Hammond Lin J Caskey C T and Chakraborty 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 P et al 2001 Validation of the AmpF STR Profiler Plus PCR Amplification Kit for use in forensic casework J Forensic Sci 46 642 646 Grossman P D Bloch W Brinson E Chang C C Eggerding F A Fung S Iovannisci D M Woo S Winn Deen E S 1994 High density multiplex detection of nucleic acid sequences oligonucleotide ligation assay and sequence coded separation Nucleic Acids Res 22 4527 4534 Grubwieser P Muhlmann R Berger B Niederstatter H Palvic M Parson W 2006 A new mini STR multiplex displaying reduced amplicon lengths for the analysis of degraded DNA Int J Legal Med 120 115 120 Hammond H Jin L Zhong Y Caskey C and Chakraborty 1994 Evaluation of 13 short tandem repeat loci for use in personal identification applications Am J Hum Genet 55 175 189 Holt C Stauffer C Wallin J et al 2000 Practical applications of genotypic Surveys for forensic STR testing Forensic Sci Int 112 91 109 Kimpton C
104. is step quickly do not save the analysis method until you finish entering settings in all of the tabs 5 After you enter settings in all tabs click Save General tab Analysis Method Editor HID settings General Allele Peak Detector Peak Quality Quality Flags Analysis Method Description Name MiniFler_AnalysisMethod_v1 Description Instrument Analysis Type HID In the Name field either type the name as shown for consistency with files supplied with other AmpF STR kits or enter a name of your choosing The Description and Instrument fields are optional 38 AmpFtSTRO MiniFiler PCR Amplification Kit User Guide Section 4 1 ID Software n Set up GeneMapper ID Software for data analysis Allele tab settings Analysis Method Editor HID General Allele Peak Detector Peak Quality Quality Flags AmpFLSTR MiniFiler 55500 Bins v1 Use marker specific stutter ratio if available Marker Repeat Type Tri Tetra Cut off Value 0 0 0 0 Minus Ratio 0 0 00 Minus Distance 00 0 0 To 00 0 0 Minus Stutter Ratio 0 0 0 0 Minus Stutter Distance From 00 3 25 00 475 Plus Stutter Ratio 0 0 0 0 Plus Stutter Distance From 00 0 0 To 00 0 0 lt 97 o 9 5 90 E E Amelogenin Cutoff Range Filter Factory D
105. les with GeneMapper ID Software 44 Examine and edit a 45 For more information a kek Rr eed rene k y eae 45 Section 4 2 GeneMapper ID X Software 48 Overview of GeneMapper ID X Software 48 Set up GeneMapper ID X Software for data analysis 49 Analyze and edit sample files with GeneMapper ID X Software 61 Examine and edita 62 more information vessi sprr Re l kas l and WE sek 45 Section 4 1 D Software Overview of D Software Instruments Before you start GeneMapper ID Software is an automated genotyping software for forensic casework databasing and paternity data analysis After electrophoresis the Data Collection Software stores information for each sample in an fsa file Using GeneMapper ID Software v3 2 1 software you can then analyze and interpret the data from the fsa files Refer to Instrument and software overview on page 16 for a list of compatible instruments When using GeneMapper ID Software v3 2 1 to perform human identification HID analysis with AmpF STR kits be aware that HID analysis requires at least one allelic ladder sample per ru
106. llele Peak Detector Peak Quality Quality Flags Peak Detection Algorithm Advanced Ranges Peak Detection Perform Analysis Sizing 3 Peak Amplitude Thresholds internal Ful Range All Sizes 50 50 validation studies to determine sa settings Smoothing and Baselining Smoothing O None Light O Heavy Peak Window Size Slope Threshold Peak Start Peak End Min Peak Half VVicth Polynomial Degree Baseline Window 51 r Size Calling Method Local Southern Method Global Southern Method Factory Defaults IMPORTANT Perform the appropriate internal validation studies to determine the peak amplitude thresholds for interpretation of data Fields include Peak amplitude thresholds The software uses these parameters to specify the minimum peak height in order to limit the number of detected peaks Although GeneMapper ID Software displays peaks that fall below the specified amplitude electropherograms the software does not label or determine the genotype of these peaks Sizecalling method This kit has been validated using the Local Southern sizing method Before using alternative sizing methods perform the appropriate internal validation studies 40 AmpFtSTRO MiniFiler PCR Amplification Kit User Guide Peak Quality tab settings AmpFtSTR MiniFiler PCR Amplification Kit User Guide Analysis Method Editor Section 4
107. log box then click OK to start analysis The status bar displays the progress of analysis as a completion bar extending to the right with the percentage indicated The table displays the row of the sample currently being analyzed in green or red if analysis failed for the sample The Analysis Summary tab is displayed and the Genotypes tab becomes available upon completion of the analysis Analysis summary window after analysis GeneMapper ID X MiniFiler Example gmidx Is Logged In Database GBOLDROYNJO9E DER File Edit Analysis View Tools Admin Help c H 2 r E B Table Setting 31XX Data Analysis E E g l Project Samples Analysis Summary Genotypes m 2 MiniFiler Example Analysis Summary C 0 2 lt D 0 p e v gt 122 e e 2 0 Select run folder to display Sample Status Total of Samples Unanalyzed 0 Analyzed 53 Analysis Setting Changed 0 Click a link below to display a filtered Samples Table containing only the samples selected Allelic Ladder Quality per run folder based SQ and CGQ only tere aeons J 8 JC 8 4 4 o 0 MiniFiler Example Control Quality per project based sample PQVs SOS SSPK MIX OMR SQ CGQ Control Type Total of Samples B All thresholds met mr One or more thresholds not met Positive Control 0 0 0 Custom Control Negative Control
108. ltiplex non nucleotide linkers are used in primer synthesis for the following loci CSF1PO FGA 0165539 018551 Amelogenin 0251338 021511 and 075820 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 AmpFtSTR MiniFiler PCR Amplification Kit User Guide 11 1 Chapter 1 Overview Product overview linkers enable reproducible positioning of the alleles to facilitate 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 eight STR loci and Amelogenin during automated DNA fragment analysis ik eu MiniFiler Kit Amplicon Length Reduction nt D7S820 129 0135317 99 021511 33 0251338 183 Amelogenin 0 018551 168 0165539 157 FGA 87 CSF1PO 201 Loci amplified by Table 1 shows the loci amplified their chromosomal locations and the corresponding the kit fluorescent marker dyes The AmpF STR MiniFiler Allelic Ladder is used to genotype the analyzed samples The alleles contained in the allelic ladder and the genotype of the AmpF STR Control DNA 007 are also listed in the table Table 1 AmpF STR MiniFiler PCR Amplification Kit loci and alleles i Ch
109. ly to interfere with accurate interpretation of mixtures Evidence samples that contain body fluids and or tissues originating from more than one individual are an integral component of forensic casework Therefore it is essential to ensure that the DNA typing system is 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 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 in the MiniFiler Kit loci in unmixed population database samples are shown in Table 8 AmpFtSTR MiniFiler PCR Amplification Kit User Guide Section 5 1 Developmental Validation Mixture studies Table 8 Peak height ratios for 0 50 ng input DNA Number of Allele Observatio Mean Median Minimum Maximum ns CSF1PO 781 87 9 89 3 57 9 100 0 0251338 911 87 0 88 8 52 3 100 0 075820 820 88 1 89 7 58 4 100 0 0135317 733 88 4 90 4 50 5 100 0 8 0165539 804 87 5 89 1 46 1 100 0 4 018551 906 87 9 89 2 55 1 100 0 5 021511 856 88 2 90 0 47 100 0
110. mance comparison hematin table 88 humic acid table 90 L Limited Product Warranty 132 LIZ size standard about 17 volume per reaction 28 29 31 loci characterization 81 chromosomal location 12 dye label 12 mapping 81 low TE buffer 19 materials and equipment 16 multicomponent analysis 15 mutation studies 95 N negative control sample preparation 21 0 off ladder alleles 69 operating systems 15 27 29 31 ordering information 117 P panel check version 47 import 48 panels import 34 part numbers 117 paternity exclusion probability of 96 PCR components validation of 66 AmpFtSTR MiniFiler PCR Amplification Kit User Guide cycle number validation 67 performing 22 setup 121 thermal cycling conditions programming 22 PCR work areas 117 121 percent stutter 75 positive control sample preparation 21 primers about 11 primers volume per reaction 21 probability of identity definition 95 Q quantification DNA 19 R reaction mix volume per reaction 21 reactions preparing for PCR 21 reagents user supplied 19 reproducibility 68 run module electrophoresis 27 29 31 5 safety biohazard 125 chemical 124 Safety Data Sheets SDSs obtaining 132 sample files fsa 34 sample preparation 21 DNA negative control 21 DNA positive control 21 size deviation sample alleles and ladder alleles 68 69 size standard create 58 sizing precision 69 species specificity 82 split peaks
111. me for the loss that occurs during reagent transfers AmpFtSTR MiniFiler PCR Amplification Kit User Guide 31 Chapter 3 Perform Electrophoresis Prepare samples for electrophoresis on the 310 instrument IMPORTANT The volume of size standard indicated in the table is a suggested amount Determine the appropriate amount of size standard based on your results and experiments 2 Pipette the required volumes of components into an appropriately sized polypropylene tube 3 Vortex the tube then centrifuge briefly 4 Into each 0 2 mL sample tube add e 25 uL of the formamide size standard mixture 1 5 uL of PCR product or allelic ladder Note For blank wells add 25 uL of Hi Di Formamide 5 Sealthe tubes with the appropriate septa then briefly centrifuge to ensure that the contents of each tube are mixed and collected at the bottom Heat the tubes in a thermal cycler for 3 minutes at 95 C Immediately place the tubes on ice for 3 minutes Place the sample tray on the autosampler Ensure that an injection list is prepared Start the electrophoresis 32 AmpFtSTR MiniFiler PCR Amplification Kit User Guide Analyze Data Section 4 1 GeneMapper ID 33 Overview of GeneMapper ID Software 33 Set up GeneMapper ID Software for data 34 Analyze and edit sample fi
112. me as shown below or enter a name of your choosing In the Size Standard Dye field select Orange In the Size Standard Table enter the sizes specified above Size Standard Editor Edit r Size Standard Description Name Description Size Standard Dye Orange r Size Standard Table Size in Basepairs 80 0 100 0 114 0 120 0 140 0 160 0 180 0 200 0 214 0 AmpFtSTR MiniFiler PCR Amplification Kit User Guide 43 Chapter 4 Analyze Data Analyze and edit sample files with GeneMapper ID Software Analyze and edit sample files with GeneMapper D Software 44 1 2 In the Project window select File Add Samples to Project then navigate to the disk or directory containing the sample files Apply analysis settings to the samples in the project The names of the settings shown are the names suggested in the sections above If you named the settings differently select the names you specified Parameter Settings Sample Type Select the sample type Analysis Method MiniFiler AnalysisMethod v1 or the name of the analysis method you created Panel MiniFiler_GS500_v1 Size Standard CE_G5_HID_GS500 or the name of the size standard you created For more information about how the Size Caller works refer to the GeneScan Analysis Software for the Windows NT Operating System Overview of the Analysis Pa
113. mend that laboratories perform internal validation studies to determine the appropriate values to use Addition of 3 A nucleotide Many DNA polymerases can catalyze the addition of a single nucleotide predominantly 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 is referred to as the A form The efficiency of A addition is related to the particular sequence of the DNA at the 3 end of the PCR product The MiniFiler Kit includes two main design features that promote maximum A addition The primer sequences have been optimized to encourage A addition The new highly robust PCR chemistry allows complete A addition with a short final incubation at 60 C for 5 minutes This final extension step gives the DNA polymerase additional time to complete A addition to all double stranded PCR products STR systems where each allele is represented by two peaks one nucleotide apart that have not been optimized for A addition may have split peaks Figure 12 shows examples of incomplete and normal A addition AmpFtSTR MiniFiler PCR Amplification Kit User Guide Section 5 1 Developmental Validation Extra peaks in the electropherogram Figure 12 Omission of the final extension step resulted in split peaks due to in
114. mples The MiniFiler Kit amplifies eight autosomal STR 0135317 075820 0251338 021511 0165539 018551 CSF1PO and FGA and the sex determining marker Amelogenin in a single PCR reaction The loci span a range between 70 to 283 nucleotides with the aid of non nucleotide linkers to achieve appropriate spacing between loci The MiniFiler Kit contains all the necessary reagents for the amplification of human genomic DNA The reagents are designed for use with the following instruments Applied Biosystems 3100 3100 Avant Genetic Analyzer e Applied Biosystems 3130 3130x Genetic Analyzer Applied Biosystems 3500 3500xL Genetic Analyzer Veriti 96 Well Thermal Cycler Part no 4375786 GeneAmp PCR System 9700 with the Silver 96 Well Block GeneAmp PCR System 9700 with the Gold plated Silver 96 Well Block The MiniFiler Kit uses primers closely flanking the STR repetitive regions miniSTRs of the DNA This amplification results in amplicons that are significantly shorter in length than those produced in the AmpFSTR Identifiler and SGM Plus PCR Amplification Kits The comparison is shown in Table 1 on page 12 Several laboratories confirm that MiniSTRs have a higher success rate for DNA analysis of degraded DNA samples Butler et al 2003 Chung et al 2004 Coble and Butler 2005 Drabek et al 2004 Grubwieser et al 2006 Wiegand et al 2001 To prevent overlap of the miniSTR amplicons in the mu
115. n GeneMapper ID X Software v1 2 for use in conjunction with data run on the Applied Biosystems 3500 Series Genetic Analyzers Normalization cannot be applied to 4 dye data so this feature is not for use with MiniFiler Kit data AmpFtSTR MiniFiler PCR Amplification Kit User Guide Section 4 2 Software Set up ID X Software for data analysis Peak Quality tab Analysis Method Editor settings Min Max Peak Height LPH MPH Homozygous min peak height Perform Heterozygous min peak height internal validation Max Peak Height studies to determine settings Peak Height Ratio PHR Min peak height ratio Broad Peak BD Max peak width basepairs Allele Number Max expected alleles Allelic Ladder Spike Cut off value Factory Defaults Save Cancel IMPORTANT Perform the appropriate internal validation studies to determine the minimum heterozygous and homozygous minimum peak height thresholds maximum peak height threshold and the minimum peak height ratio threshold for interpretation of MiniFiler Kit data AmpFtSTR MiniFiler PCR Amplification Kit User Guide 57 C 0 2 0 lt D 0 e v gt 122 e e 2 0 4 Chapter 4 GeneMapper ID X Software Set up GeneMapper ID X Software for data analysis SQ amp GQ tab
116. n folder Perform the appropriate internal validation studies if you want to use multiple ladder samples in an analysis For multiple ladder samples the GeneMapper ID Software calculates allelic bin offsets by using an average of all ladders that use the same panel within a run folder Allelic ladder samples in an individual run folder are considered to be from a single run AmpFtSTR MiniFiler PCR Amplification Kit User Guide 33 4 Chapter 4 Analyze Data Set up GeneMapper ID Software for data analysis When the software imports multiple run folders into a project only the ladder s within their respective run folders are used for calculating allelic bin offsets and subsequent genotyping Allelic ladder samples must be labeled as Allelic Ladder in the Sample Type column in a project Failure to apply this setting for ladder samples results in failed analysis Injections containing the allelic ladder must be analyzed with the same analysis method and parameter values that are used for samples to ensure proper allele calling Alleles that are not in the AmpF STR Allelic Ladders do exist Off ladder OL alleles may contain full and or partial repeat units An off ladder allele is an allele that occurs outside the 0 5 nt bin window of any known allelic ladder allele or virtual bin Note If a sample allele peak is called as an off ladder allele verify the sample result according to your laboratory s protocol Set
117. ng 1993 e mutation studies Hammond et al 1994 Brinkmann et al 1995 Chakraborty et al 1996 Chakraborty et S al 1997 Brinkmann et al 1998 Momhinweg et al 1998 Szibor et al 1998 of direct mutation rate counts produced Larger sample sizes for some of the MiniFiler 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 Probability of identity Table 10 shows the Probability of identity values of the MiniFiler Kit loci individually and combined The Pj value is the probability that two individuals selected at random will have an identical MiniFiler Kit genotype Sensabaugh 1982 The Pr values for the populations described in this section are then approximately 1 1 53 X 10 10 African American 1 1 22 X 10 10 U S Caucasian 1 9 57 X 10 9 U S Hispanic and 1 4 82 X 10 Native American Table 10 Probability of identity Pj values for the MiniFiler Kit loci Locus African Caucasian Hispanic Nany American American CSF1PO 0 079 0 132 0 141 0 123 0251338 0 023 0 027 0 038 0 043 075820 0 085 0 063 0 083 0 081 0135317 0 132 0 079 0 056 0 056 0165539 0 077 0 097 0 090 0 082 018551 0 033 0 031 0 031 0 046 021511 0 037 0 044 0 047 0 074 0 034 0 035 0 032 0 031 Combined 6 52 x 1
118. niFiler Kit AmpFtSTR9 MiniFiler PCR Amplification Kit User Guide 105 5 Chapter 5 Performance Validation After Buffer and Enzyme Component Replacement Degraded DNA study Figure 28 Degraded DNA study intracolor balance 0 5 ng input DNA amount Control Control B 4 Test A Test Test Intracolor Balance Mean Panel variable Sample Mean referenced Overall mean referenced peak height observations were consistent between all Test peak height and Control mixes with the exception of Test C Mix on pristine DNA which showed slightly higher referenced peak heights overall The mean referenced peak height results for all Test and Control mixes fall within the expected range of performance for the MiniFiler Kit Figure 29 Degraded DNA study intracolor balance 0 5 ng input DNA amount DEG 007 PRI 007 Lot Control Control B 4 Test A Peak Height Mean 106 AmpFtSTR MiniFiler PCR Amplification Kit User Guide Allelic dropout Section 5 2 Performance Validation After Buffer and Enzyme Component Replacement Degraded DNA study Simulated degraded samples showed an overall drop in peak height compared to pristine DNA samples Peak height of the higher molecular weight loci showed a greater drop in peak height compared to lower molecular weight loci in some cases leading to allelic dropout This is representative of the typical
119. nor component The profiles of the samples in Figure 19 are shown in Table 9 92 AmpFtSTR MiniFiler PCR Amplification Kit User Guide Section 5 1 Developmental Validation Population data Table 9 Genotypes of mixed DNA samples Allele lesa DN DD Profile Sample B D135317 11 12 14 075820 7 12 8 9 Amelogenin X Y X Y D251338 20 23 20 21 021511 28 31 28 30 0145529 9 10 12 13 018551 12 15 17 19 CSF1P0 11 12 10 FGA 24 26 21 22 MiniFiler kit has been optimized to amplify and type approximately 0 50 to 0 75 ng of single source DNA reliably Population data SWGDAM guideline The distribution of genetic markers in populations should be determined in relevant 27 population groups SWGDAM July 2003 Overview 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 e 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 The same as the genotype of a suspects reference sample 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 estimate
120. ns and marker stutter 48 Create an analysis method 53 General tab 5 54 Alleletab SettingS LEER edd etg 55 Peak Detector tab settings 56 AmpFtSTR MiniFiler PCR Amplification Kit User Guide Contents Peak Quality tab settings 57 SQ S DN DN aee Sep Sheet Show teed whe RS 58 Create size standard optional 58 Analyze and edit sample files with GeneMapper D X Software 60 Examine and edit a project 2 2 2 61 For more informatlon i ud xu Xa cd cae eevee ee eam pee eL Cete DR etm 62 CHAPTER5 Experiments and Results 63 Section 5 1 Developmental Validation 65 a E EN EE E EE 65 Experiments using the 65 imp rance di validation zee gt yan keka Ee E ec eed eee ae A 65 Ey AE QUEU
121. nstrument and software overview on page 16 for a list of compatible instruments Before you start When using GeneMapper ID X Software v1 0 1 or higher to perform human identification HID analysis with AmpF STR kits be aware that 46 HID analysis requires at least one allelic ladder sample per run folder Your laboratory can use multiple ladder samples in an analysis provided individual laboratories conduct the appropriate validation studies For multiple ladder samples the GeneMapper ID X Software calculates allelic bin offsets by using an average of all ladders that use the same panel within a run folder Allelic ladder samples in an individual run folder are considered to be from a single run When the software imports multiple run folders into a project only the ladder s within their respective run folders are used for calculating allelic bin offsets and subsequent genotyping Allelic ladder samples must be labeled as Allelic Ladder in the Sample Type column in a project Failure to apply this setting for ladder samples results in failed analysis Injections containing the allelic ladder must be analyzed with the same analysis method and parameter values that are used for samples to ensure proper allele calling Alleles that are not in the AmpF STR Allelic Ladders do exist Off ladder OL alleles may contain full and or partial repeat units An off ladder allele is an allele that occurs outside the 0 5 nt bin win
122. ntains high concentrations of a PCR inhibitor for example heme compounds certain dyes Quantitate DNA and add minimum necessary volume Repeat test Wash the sample in a Centricon 100 114 AmpFtSTR MiniFiler PCR Amplification Kit User Guide The 3rd Order Least Squares Sizing Method You can use the 3rd Order Least Squares Sizing Method as an alternative to the Local Southern method when analyzing MiniFiler Kit data When to use We recommend using the 3rd Order Least Squares method to size data obtained when analyzing MiniFiler Kit data using the GeneScan 500 LIZ Size Standard The Least Squares methods both 2nd Order and 3rd Order use regression analysis to build a best fit size calling curve This method is used to extrapolate sizes that extend beyond the physical range of the size standard Small lt 75 nt fragments generated by the MiniFiler Kit can be sized using the GeneScan 500 LIZ Size Standard About the Local Southern algorithm For the highest level of accuracy the Local Southern method requires two size standard fragments below the smallest unknown fragment and two size standard fragments above the largest unknown fragment All AmpF STR kits except the MiniFiler Kit have an allele size range between 100 to 360 nt Optimal genotyping accuracy of all kits other than MiniFiler require detection of all of the GeneScan 500 LIZ Size Standard or ROX fragments between
123. o 0 25 Genotype Quality From 0 75 to 1 0 From 0 0440 0 25 Factory Defaults IMPORTANT The values shown are the software defaults and are the values we used during developmental validation Perform the appropriate internal validation studies to determine the appropriate values to use in your laboratory 42 AmpFtSTRO MiniFiler PCR Amplification Kit User Guide Section 4 1 ID Software n Set up GeneMapper ID Software for data analysis Create a size The GeneScan 600 LIZ9 Size Standard v2 0 contains the following size standard standard peaks GeneScan 600 1129 Size Standard v2 0 peak sizes 60 80 100 114 120 140 160 180 200 214 220 240 250 260 280 300 314 320 340 360 380 400 414 420 440 and 460 To create the size standard definition file 9 5 1 Select Tools GeneMapper Manager to open the GeneMapper Manager lt D GeneMapper ID X Manager y o Find Name Containing o L 9 B iS Projects Analysis Methods Table Settings Plot Settings Matrices Size Standards Report Settings Last Saved Owner Type Description CE F HID 5500 75 400 2007 08 09 13 23 gmidx Advanced HID 65500 75 450 2007 08 09 13 24 C gmidx Advanced G5 HID 25500 2011 04 18 13 15 gmidx Advanced 2 Select the Size Standards tab then click New 3 Enter a na
124. oduct Description Quantifiler Human DNA Quantification Kit Part no 4343895 and Quantifiler9 Y Human Male DNA Quantification Kit Part no 4343906 For more information see Quantifiler Human DNA Quantification Kits User s Manual Pub no 4344790 Properties The Quantifiler Human and Quantifiler Y Human Male Kits are highly specific for human DNA and they individually detect total human or male DNA respectively The kits detect single stranded and degraded DNA How they work The Quantifiler DNA Quantification Kits consist of target specific and internal control 5 nuclease assays The Quantifiler Human and Quantifiler Y Human Male Kits contain different target specific assays human DNA or human male DNA respectively that each consist of two locus specific PCR primers and one TaqMan MGB probe labeled with FAM dye for detecting the amplified sequence The kits each contain a separate internal 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 and one TaqMan probe labeled with VIC dye for detecting the amplified IPC Quantifiler Duo DNA Quantification Kit Part no 4387746 For more information see Quantifiler Duo DNA Quantification Kit User s Manual Pub 4391294 lt The Quantifiler Duo Kit is highly specific for human This kit combines
125. on etc To obtain SDSs see the Documentation and Support section in this document AmpFtSTR9 MiniFiler PCR Amplification Kit User Guide 123 Appendix E Safety Chemical safety Chemical safety A WARNING GENERAL CHEMICAL HANDLING To minimize hazards ensure laboratory personnel read and practice the general safety guidelines for chemical usage storage and waste provided below and consult the relevant SDS for specific precautions and instructions Read and understand the Safety Data Sheets 5056 provided by the chemical manufacturer before you store handle or work with any chemicals or hazardous materials To obtain SDSs see the Documentation and Support section in this document Minimize contact with chemicals Wear appropriate personal protective equipment when handling chemicals for example safety glasses gloves or protective clothing Minimize the inhalation of chemicals Do not leave chemical containers open Use only with adequate ventilation for example fume hood Check regularly for chemical leaks or spills If a leak or spill occurs follow the manufacturer s cleanup procedures as recommended in the SDS Handle chemical wastes in a fume hood Ensure use of 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
126. ork area IMPORTANT Place the thermal cyclers in the Amplified DNA Work Area You can use the following systems Veriti 96 Well Thermal Cycler Part no 4375786 e GeneAmp PCR System 9700 with the Silver 96 Well Block GeneAmp PCR System 9700 with the Gold plated Silver 96 Well Block IMPORTANT The MiniFiler Kit is validated for use with the Veriti 96 well Thermal Cycler Part no 4375786 and the GeneAmp PCR System 9700 with the silver block Part no N8050251 or the gold plated silver block Part no 4314443 It is not verified for use with the Veriti 96 Well Fast Thermal Cycler Part no 4375305 or the GeneAmp PCR System 9700 with the aluminium block Part no 4314879 122 AmpFtSTR MiniFiler PCR Amplification Kit User Guide Safety WARNING GENERAL SAFETY Using this product in a manner not specified the user documentation may result in personal injury or damage to the instrument or device Ensure that anyone using this product has received instructions in general safety practices for laboratories and the safety information provided in this document Before using an instrument or device read and understand the safety information provided in the user documentation provided by the manufacturer of the instrument or device Before handling chemicals read and understand all applicable Safety Data Sheets SDSs and use appropriate personal protective equipment gloves gowns eye protecti
127. ormance of the three kits in a simulated model of humic acid inhibition Only those loci gt 50 RFU represented in the MiniFiler Kit were measured in the Identifiler Kit and SGM Plus Kit see Table 7 A complete profile with control 007 DNA yields 17 peaks using the MiniFiler Kit AmpFtSTR9 MiniFiler PCR Amplification Kit User Guide 89 Mixture studies Chapter 5 Experiments and Results Table 7 Comparison of MiniFiler Identifiler and SGM Plus Kit performance in simulated model of humic acid inhibition n 5 Humic Acid MiniFiler Kit Identifiler Kit SGM Plus kit 10 ng uL 17 17 17 17 17 17 17 17 17 17 17 17 14 14 14 14 14 14 17 17 17 17 17 17 14 17 14 14 14 14 30 ng uL 17 17 17 17 17 17 0 17 0 17 0 17 0 0 14 0 14 0 14 0 17 17 17 17 17 0 17 14 0 14 50 ng uL 17 17 17 17 17 17 0 17 0 17 0 17 0 0 14 0 14 0 14 0 17 17 14 17 17 0 17 14 0 14 Mixture studies SWGDAM guideline 2 8 Mixture studies 90 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 the results We recommend that individual laboratories assign a minimum peak height threshold based on validation experiments performed in each laboratory to avoid typing when stochastic effects are like
128. p 8 Tube Strip 0 2 mL N8010580 MicroAmp 8 Strip N8010535 MicroAmp 96 Well Tray Retainer Set 403081 MicroAmp 96 Well Base N8010531 MicroAmp Clear Adhesive Film 4306311 MicroAmp Optical Adhesive Film 4311971 MicroAmp Optical 96 Well Reaction Plate N8010560 Other user supplied materials Hi Di Formamide 25 mL 4311320 Aerosol resistant pipette tips MLS Microcentrifuge tubes MLS Pipettors MLS Tape labeling MLS Tube 50 mL Falcon MLS Tube decapper autoclavable MLS Deionized water PCR grade MLS AmpFtSTR MiniFiler PCR Amplification Kit User Guide 119 Appendix C Ordering Information Equipment and materials not included Source Vortex MLS For the Safety Data Sheet SDS of any chemical not distributed by Life Technologies contact the chemical manufacturer Before handling any chemicals refer to the SDS provided by the manufacturer and observe all relevant precautions 120 AmpFtSTR MiniFiler PCR Amplification Kit User Guide PCR Work Areas Work area setup and lab 121 PCR setup work 121 Amplified DNA work 2 2 2 122 Work area setup and lab design Many resources are available for the appropriate design of a PCR laboratory If you are using an AmpF STR Kit for Forensic DNA testing refer to For
129. pattern observed in partially degraded samples Profile morphology and levels of allelic dropout were comparable across all Test and Control mixes demonstrating equivalent performance Degraded DNA study summary of Allelic Dropout observed at 125 pg input DNA concentration Number of Number of Number of Percent of Reagent Mix samples alleles alleles alleles P expected dropped dropped Control A 5 85 3 4 Control B 5 85 9 11 Test A 5 85 12 14 Test B 5 85 5 6 Test 5 85 13 15 Figure 30 Degraded DNA study representative electropherograms from 500 pg input DNA amplifications of simulated degraded DNA samples Y scale 1000 RFU 65 85 1000 105 125 145 165 185 205 225 245 00 65 85 1000 105 125 145 165 185 205 z 225 0 Sample for Deletion 245 800 85 85 1000 105 125 145 165 185 205 j Control 225 Mark Sample for Deletion 245 200 s00 55 100 205 ps Test A Mark Sample for Deletion 245 s00 E 0 55 85 1000 105 125 145 165 185 205 225 Mark Sample for Deletion 245 200 y Test AmpFtSTR MiniFiler PCR Amplification Kit User Guide 107 U D ED 3 3 hy 2 e 0 5 gel 2 00 e ra 2 oH m N lt 3 9 2 9 Em 5
130. presents 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 68 AmpFtSTR MiniFiler PCR Amplification Kit User Guide Section 5 1 Developmental Validation Accuracy precision and reproducibility Figure 7 Allele Size vs Allelic Ladder Sizing for 42 samples analyzed on an Applied Biosystems 3130xl Genetic Analyzer 0 75 o CSF1PO FGA 018551 0165539 D24811 0138317 075820 0 25 NESE EOE S 9 M 1 8 wt ue Boz T eu Ma z 9 og By 0 Q o 5 0 00 ve B r q e bad H rt xd jj rz 3 2 1 2 244 a i gt NY ir A aa at a g o tet ge Er lt Ot 8 tat ig i o amp E 8 e l y S TEN gt 157465705 622 Ee We cc jg o wa JR Re DEN S RAN r o o amp 4 catia mm 0 75 FHTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT 80 100 120 140 160 180 200 220 240 260 280 Allele Size nt Precision and size Sizing precision enables the determination of accurate and reliable genotypes Sizing windows precision was measured on an Applied Biosystems
131. put Amount Reagent Mix Genotype Concordance 2 125 0 Test 100 8 Test B 100 8 5 100 amp 100 3 Control B 100 0 250 pg Test 100 E Test B 100 Test 100 a Control A 100 Control 100 2 500 Test 100 8 Test 100 2 Test C 100 Control 100 Control 100 750 pg Test 100 Test 100 Test 100 Control 100 Control 100 Degraded DNA study To reflect the specific design of the MiniFiler Kit for degraded samples 5 replicates of 0 5 ng degraded Control DNA 007 DNA and 5 replicates of 0 5 ng pristine Control DNA 007 were amplified Results were evaluated for intracolor balance mean referenced peak height and levels of allelic dropout degraded DNA replicates only Degraded DNA was prepared by first sonicating the DNA then treating with 1 U DNAse I enzyme for increasing time increments to simulate increasing levels of degradation A final input DNA concentration of 500 pg was used for all amplifications Intracolor balance No significant difference 1076 increase of decrease in the level of intracolor balance was observed between the Test and Control mixes on either degraded or pristine DNA with the exception of Test A Mix which showed higher levels of intracolor balance for the NED yellow dye in degraded samples The levels of intracolor balance obtained for all Test and Control mixes fall within the expected range of performance for the Mi
132. r ID X Software for data analysis 3 Check the version of files imported into the Panel Manager Panel Manager File Edit Bins View Sain mw Panel Manager a Select Panel Manager in the navigation pane b Expand the Panel Manager folder and any sub folders to identify the analysis file version already installed for your kit choice 4 Check the version of files available for import into the Panel Manager a Select Panel Manager then select File Import Panels to open the Import Panels dialog box b Navigate to then open the Panels folder and check the version of panel bin and stutter files installed 5 versions are available on the website download and import described below Import panels To import the MiniFiler Kit panel bin set and marker stutter from our web site into bins and marker the GeneMapper ID X Software database stutter 1 Download and open the file containing panels bins and marker stutter a Goto www lifetechnologies com support gt Software Patches amp Updates GeneMapper ID X Software Download the file AmpFLSTR Analysis Files GMIDX b Unzip the file 2 Start the GeneMapper ID X Software then log in with the appropriate user name and password IMPORTANT For logon instructions refer to the GeneMapper ID X Software Version 1 0 Getting Started Guide Pub no 4375574 3 Select Tools Panel Manag
133. r J and Wong C 1993 Mutation of human short tandem repeats Hum Mol Genet 2 1123 1128 Weir B 1990 Genetic Data Analysis Sinauer Associates Sunderland MA AmpFtSTR MiniFiler PCR Amplification Kit User Guide 129 Bibliography Wiegand P and Kleiber M 2001 Less is more length reduction of STR amplicons using redesigned primers Int J Legal 114 285 287 130 AmpFtSTR MiniFiler PCR Amplification Kit User Guide Documentation and Support Related documentation Document title um 3100 3100 Avant Data Collection v2 0 User Guide 4347102 3100 3100 Avant Genetic Analyzers Using Data Collection Software v2 0 User Bulletin 4350218 3100 Genetic Analyzer User Manual Data Collection v1 1 4315834 3100 3100 Avant Genetic Analyzers Protocols for Processing AmpF STR9 PCR Amplification Kit PCR Products 4332345 User Bulletin Applied Biosystems 3130 3100xl Genetic Analyzers Using Data Collection Software v3 0 User Bulletin 4363787 Applied Biosystems 3130 3130xl Genetic Analyzers Getting Started Guide 4352715 Applied Biosystems 3130 3130xl Genetic Analyzers Maintenance Troubleshooting and Reference Guide 4352716 Applied Biosystems 3130 3130xl Genetic Analyzers Quick Reference Card 4362825 Applied Biosystems 3130 3130xl Genetic Analyzers AB Navigator Software Administrator Guide 4359472 Applied Biosystems 3130 3100xl DNA Analyzers User Guide 4331468 Applied Biosystems 3500 3500x
134. rameters and Size Caller User Bulletin Part no 4335617 For additional information about size standards refer to the GeneMapper ID Software Version 3 1 Human Identification Analysis User Guide Part no 4338775 Click j Analyze enter a name for the project in the Save Project dialog box then click OK to start analysis During analysis status bar displays the progress of analysis as both Acompletion bar extending to the right with the percentage completed indicated Text messages on the left Thetable displays the row of the sample currently being analyzed in green or red if analysis failed for the sample Genotypes tab becomes available after analysis AmpFtSTR MiniFiler PCR Amplification Kit User Guide Section 4 1 ID Software Examine and edit a project The following figure shows the analysis summary window after analysis 2 GeneMapper ID v3 2 1 Untitled gmid Is Logged In File Edit Analysis View Tools Help Ec 0 BEA mH zl Table Setting AmpFLSTR Table E GjProject LMiniFiler Example Samples Genotypes Status Sample File Sample Type Analysis Method Panel Allelic Ladder tsa Allelic Ladder MiniFiler amp nalysisMetho MiniFiler GS500 v1 Sample 1 fsa Sample MiniFiler amp nalysisMetho MiniFiler GS500 v1 Sample 10 fsa Sample MiniFiler amp nalysisMetho MiniFiler 55500 v1
135. re Download the file GMID MiniFiler files b Unzip the file AmpFtSTR MiniFiler PCR Amplification Kit User Guide Section 4 1 ID Software n Set up GeneMapper ID Software for data analysis 2 Start the GeneMapper ID Software then log in with the appropriate user name and password IMPORTANT For logon instructions refer to the GeneMapper ID Software Version 3 1 Human Identification Analysis User Guide Part no 4338775 3 Select Tools Panel Manager 4 Find then open the folder containing the panels and bins a Select Panel Manager in the navigation pane Panel Manager b Select File Import Panels to open the P ee Dr Mah Import Panels dialog box A li Navigate to then open the MiniFiler Panel Manager Analysis Files GMID folder that you unzipped in step 1 on page 34 lt 97 9 5 90 E 5 Select AmpFLSTR_MiniFiler_GS500_Panels_v1 txt then click Import Note Importing this file creates a new folder in the navigation pane of the Panel Manager AmpFISTR_MiniFiler_GS500_Panels_v1 This folder contains the panel and associated markers Import Panels Look in MiniFiler Analysis Files E E E AmpFLSTR_MiniFiler_GS500_Bins_v1 txt J AmpFLSTR MiniFiler GS500 Panels_v1 txt My Recent MiniFiler 55500 HID v1 xml Documents 2 Desktop File
136. romosome s Dye Control Locus designation location Alleles included in Allelic Ladder label DNA 007 0135317 13422 31 8 9 10 11 12 13 14 15 6 FAM 11 D7S820 7411 21 22 6 7 8 9 10 11 12 13 14 15 7 12 Amelogenin X p22 1 22 3 X Y vic X Y Y p11 2 0251338 2435 37 15 16 17 18 19 20 21 22 23 24 25 26 20 23 27 28 021511 21411 2 421 24 24 2 25 26 27 28 28 2 29 29 2 30 28 31 30 2 31 31 2 32 32 2 33 33 2 34 34 2 35 35 2 36 37 38 0165539 16q24 qter 5 8 9 10 11 12 13 14 15 NED 9 10 018551 18421 3 7 9 10 10 2 11 12 13 13 2 14 14 2 15 12 15 16 17 18 19 20 21 22 23 24 25 26 27 CSF1PO 9q33 3 34 6 7 8 9 10 11 12 13 14 15 PET 11 12 FGA 4q28 17 18 19 20 21 22 23 24 25 26 26 2 27 24 26 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 12 AmpFtSTR MiniFiler PCR Amplification Kit User Guide Chapter 1 Overview 1 Product overview Allelic ladder Figure 1 shows the allelic ladder for the MiniFiler Kit See Allelic ladder requirements on page 26 for information on ensuring accurate genotyping Figure 1 GeneMapper D X Software plot of the AmpF STR MiniFiler Allelic Ladder 100 110 120 130 140 20 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 70 80 90 1800 1200 600 0 2 m a fas ffs 7 lel M 5 AME
137. rt of the Committee on DNA Forensic Science National Research Council 1996 These databases are summarized in Chapter 5 of the AmpFtSTR Identifiler PCR Amplification Kit User Guide Part no 4323291 The minimum reportable genotype frequency at each locus is as follows 1 19 x 10 4 for the African American database 1 19 x 10 4 for the U S Caucasian database e 170 x 10 4 for the U S Hispanic database and e 2 97 x 10 4 for the Native American database p p 1 p where 0 01 Therefore the minimum combined multilocus genotype frequency at 8 loci is as follows 4 02 x 10 32 for the African American database 4 02 x 10 32 for the U S Caucasian database e 6 98 x 10 for the U S Hispanic database and 6 05 x 10 29 for the Native American database Primer relocation in the MiniFiler Kit could unintentionally lead to allele imbalance or allele dropouts that are not found in the Identifiler kit These may be caused by a SNP or a deletion in the primer binding site Experimental data was used to quantitate allele calling differences between the MiniFiler Kit and the Identifiler Kit We analyzed 1 064 samples 353 Caucasians 347 African Americans 207 Hispanics and 157 Asians by comparing allele calls between the Identifiler Kit and MiniFiler Kit In the majority of samples analyzed the results were found to be concordant between the kits with minor discordancy found in few exceptions Discordant data
138. s basic characteristics of the eight loci and the sex determining marker Amelogenin which are amplified with the MiniFiler Kit Most of these loci have been extensively characterized by other laboratories The primers for the Amelogenin locus flank a six nucleotide deletion within intron 1 of the X homologue Amplification results in 104 nt and 110 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 MiniFiler 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 We have sequenced all the alleles in the AmpF STR MiniFiler Allelic Ladder In addition other groups in the scientific community have sequenced alleles at some of these loci Among the various sources of sequence data on the MiniFiler 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 inheritance of various DNA loci Each family set contains three generations generally including four grandparents two parents and several offspring Consequently the
139. separately e AmpE STR MiniFiler Allelic Ladder Developed for accurate characterization of the alleles amplified by the MiniFiler Kit The Allelic Ladder contains most of the alleles reported for the 16 autosomal loci See page 12 for a list of the alleles included in the Allelic Ladder AmpFtSTR MiniFiler PCR Amplification Kit User Guide 17 1 Chapter 1 Overview Materials and equipment 18 AmpFtSTR MiniFiler PCR Amplification Kit User Guide Required user supplied 19 a DNA quantification ccs sisi toe K KA Ne YO RR K k 19 Prepare the amplification kit reactions 21 Perform PCR hate ertet E eU 4 e oe p Le e ih 22 E Amplification using bloodstained FTA cards 22 Required user supplied reagents In addition to the Identifiler Kit reagents the use of low TE buffer 10 mM Tris 0 1 mM EDTA pH 8 0 is recommended You can prepare the buffer as described in the procedure below or order it from Teknova Cat T0223 To prepare low TE buffer 1 Mix together l0mL of 1 M Tris HCl pH 8 0 02 mL of 0 5 M EDTA pH 8 0 990 mL glass distilled or deionized water Note Adjust the volumes accordingly for specific needs 2 Aliquot and autoclave the solutions 3 Store at room temperature DNA quantification Importance of
140. ser Guide Bibliography Momhinweg E Luckenbach C Fimmers R and Ritter H 1998 D351358 sequence analysis and gene frequency in 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 5 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 gov hq lab fsc current standards 2004_03_standards02 htm Sensabaugh G F 1982 Biochemical markers of individuality In Saferstein R ed Forensic Science Handb ook Prentice Hall Inc New York pp 338 415 Sharma V and Litt M 1992 Tetranucleotide repeat polymorphism at the 021511 locus Hum Mol Genet 1 67 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
141. st 4 U D ED 3 3 hy 2 e 0 5 gel 2 00 e ra 2 oH m N lt 3 9 2 9 Em 5 e D 3 D 5 N m Q E E E 125 250 500 750 125 250 500 750 DNA Concentration pg Panel variable Sample AmpFtSTR MiniFiler PCR Amplification Kit User Guide 101 Chapter 5 Performance Validation After Buffer and Enzyme Component Replacement Sensitivity study Figure 24 Sensitivity study representative electropherograms for Sample 2 amplified using 250 pg input DNA Y scale 500 RFU 30 110 430 150 170 490 210 230 800 400 200 Control A Mark Sample for Deletion 410 130 150 170 130 210 230 800 600 400 200 Control B Mark Sample for Deletion 110 130 150 170 190 210 230 600 500 300 200 100 Test A Mark Sample for Deletion 410 130 150 170 130 210 230 600 200 Test B Mark Sample For Deletion 110 130 150 170 190 210 230 600 400 200 Test C DNA concentration and peak height 102 The calculated slope and R values for each of the plotted curves are equivalent showing comparable relationships between peak height and DNA input amount for the Test and Control mixes Figure 25 AmpFtSTR MiniFiler PCR Amplification Kit User Guide Section 5 2 Performance Validation After Buf
142. standard deviations 668 are shown in Table 4 on page 78 Peaks in the stutter position that are above the stutter filter percentage specified in the software are not filtered Peaks in the stutter position that have not been filtered and remain labeled can be further evaluated The measurement of stutter percentage for allele peaks that are off scale may be unusually high AmpFtSTR MiniFiler PCR Amplification Kit User Guide 75 g lt o e 3 o e 5 E s 2 76 Chapter 5 Experiments and Results Extra peaks in the electropherogram Figure 8 Stutter percentages for 0135317 075820 loci Percent Stutter em o Si J hiri E aou XW 8 10 11 12 13 14 0135317 15 16 5 6 7 8 g 10 11 12 13 14 15 075820 Figure 9 Stutter percentages for the 0251338 021517 loci Percent Stutter 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 0251338 021511 AmpFtSTR MiniFiler PCR Amplification Kit User Guide Section 5 1 Developmental Validation Extra peaks in the electropherogram Figure 10 Stutter percentages for the D16S539 018551 loci 18 17 16 15 5218 14 x 13 t i i 222 i 12 1 211415 T i lt 11 i 4 e 10
143. 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 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 stutter percent is measured by dividing the height of the stutter peak by the height of the main allele peak Peak heights were measured for amplified samples n 967 at the loci used in the MiniFiler Kit data were generated on the Applied Biosystems 3130x Genetic Analyzer Some conclusions from these measurements and observations are e For each MiniFiler Kit locus the stutter percentage generally increases with allele length as shown in Figure 8 on page 76 through Figure 11 on page 77 Smaller alleles display a lower level of stutter relative to the longer alleles within each locus Each allele within a locus displays a consistent stutter percentage Stutter filter sets in GeneMapper ID and GeneMapper ID X Software calculated as the mean stutter for the locus plus three
144. t e ted CER Re 29 Electrophoresis software setup and reference lt 29 Prepare samples for electrophoresis on the 3500 3500xL instruments 29 Section 3 3 310 Instr AT 2 a rtr esp E rur wale ER 31 Set up the 310 instrument for electrophoresis 31 Reagents and parts 31 Electrophoresis software setup and reference lt 31 Prepare samples for electrophoresis on the 310 31 CHAPTER 4 Analyze Data 33 Section 4 1 GeneMapper ID Software 33 Overview of GeneMapper ID Software 33 InStr rnlents zer Re e 33 Beforeyou start REIR Neb MIA REESE MAS 33 Set up GeneMapper ID Software for data 34 trs Son ftu M S o 34 Before using the software for the 34 Import panels and bins 34 Create an analysis method 38 General tab 5
145. tSTR MiniFiler PCR Amplification Kit User Guide 71 g lt e 3 e 5 5 5 e 72 Chapter 5 Experiments and Results Accuracy precision and reproducibility Allele Mean Standard Deviation 21 179 93 179 96 0 045 0 055 22 183 92 183 95 0 048 0 064 23 187 89 187 94 0 043 0 062 24 191 87 191 91 0 045 0 058 018551 7 124 68 124 73 0 035 0 060 9 132 53 132 57 0 044 0 059 10 136 46 136 50 0 040 0 056 10 2 138 37 138 42 0 040 0 056 11 140 38 140 43 0 038 0 055 12 144 33 144 37 0 039 0 059 13 148 27 148 31 0 048 0 054 13 2 150 19 150 22 0 040 0 062 14 152 21 152 24 0 043 0 057 14 2 154 14 154 18 0 035 0 054 15 156 17 156 20 0 042 0 061 16 160 13 160 16 0 047 0 060 17 164 06 164 10 0 046 0 057 18 168 05 168 06 0 039 0 058 19 172 00 172 02 0 041 0 054 20 175 97 175 99 0 035 0 061 21 179 93 179 96 0 045 0 055 22 183 92 183 95 0 048 0 064 23 187 89 187 94 0 043 0 062 24 191 87 191 91 0 045 0 058 25 195 85 195 87 0 053 0 070 26 199 86 199 89 0 047 0 063 27 203 84 203 88 0 044 0 070 021511 24 186 96 186 99 0 056 0 067 24 2 188 95 188 97 0 047 0 066 25 190 89 190 92 0 051 0 071 26 194 82 194 84 0 052 0 063 27 198 67 198 7 0 053 0 071 28 202 71 202 74 0 055 0 065 28 2 204 63 204 66 0 060 0 070 29 206 73 206 77 0 048 0 072 29 2 208 50 208 55 0 051 0 077 AmpFtSTR Min
146. te an analysis Use the following procedure to create an analysis method for the SGM Plus Kit method IMPORTANT Analysis methods are version specific so you must create an analysis method for each version of the software For example an analysis method created for GeneMapper ID X version 1 2 is not compatible with earlier versions of GeneMapper ID X Software or with GeneMapper ID Software version 3 2 1 1 Select Tools GeneMapper ID X Manager to open the GeneMapper ID X Manager GeneMapper ID X Manager Find Name Containing C 0 2 0 lt i 0 p e v gt 122 e p 2 0 mM Projects Analysis Methods Table Settings Plot Settings Matrices Size Standards Report Settings 2 Select the Analysis Methods tab then click New to open the Analysis Method Editor with the General tab selected The figures below show the settings for each tab of the Analysis Method Editor Configure the Analysis Method Editor tab settings as shown in the figures below unless the instructions state otherwise Note The Analysis Method Editor closes when you save your settings To complete this step quickly do not save the analysis method until you finish entering settings in all of the tabs 3 After you enter settings in all tabs click Save AmpFtSTR9 MiniFiler PCR Amplification Kit User Guide 53 4 Chapter 4 GeneMapper ID X Software Set up GeneMapp
147. ted in the table is a suggested amount Determine the appropriate amount of size standard based on your experiments and results AmpFtSTR MiniFiler PCR Amplification Kit User Guide 29 Chapter 3 3500 3500xL Instruments Prepare samples for electrophoresis on the 3500 3500xL instruments 2 Pipet the required volumes of components into an appropriately sized polypropylene tube 3 Vortex the tube then centrifuge briefly 4 Into each well of a MicroAmp Optical 96 Well Reaction Plate add 9uL of the formamide size standard mixture 1 of PCR product or Allelic Ladder Note For blank wells add 10 uL of Hi Di Formamide 5 Sealthe reaction plate with appropriate septa then briefly vortex and centrifuge the plate to ensure that the contents of each well are mixed and collected at the bottom Heat the plate in a thermal cycler for 3 minutes at 95 C Immediately place the plate on ice for 3 minutes Place the sample tray on the autosampler or Woe we Start the electrophoresis run 30 AmpFtSTR MiniFiler PCR Amplification Kit User Guide Chapter 3 Perform Electrophoresis Set up the 310 instrument for electrophoresis Section 3 3 310 Instrument Set up the 310 instrument for electrophoresis Reagents and parts Ordering Information on page 117 lists the required materials not supplied with the AmpF STR Identifiler PCR Amplification Kit IMPORTANT The fluorescent dyes attached to the prim
148. tho MiniFiler GS500 v1 C D ied o 19 o 122 e 2 edit project You can display electropherogram plots from the Samples and Genotypes tabs of the Project window to examine the data These procedures start with the Samples tab of the Project window assuming the analysis is complete For more information For details about GeneMapper ID Software features allele filters peak detection algorithms and project editing refer to GeneMapper ID Software Versions 3 1 and 3 2 Human Identification Analysis Tutorial Part no 4335523 e GeneMapper ID Software Version 3 1 Human Identification Analysis User Guide Part no 4338775 Installation Procedures and New Features for GeneMapper ID Software Software Version v3 2 User Bulletin Part no 4352543 AmpFtSTR MiniFiler PCR Amplification Kit User Guide 45 n Chapter 4 GeneMapper ID X Software Overview of GeneMapper ID X Software Section 4 2 GeneMapper D X Software Overview of GeneMapper D X Software GeneMapper ID X Software is an automated genotyping software for forensic casework databasing and paternity data analysis After electrophoresis the data collection software stores information for each sample in a fsa or hid file Using GeneMapper ID X Software v1 0 1 or higher you can then analyze and interpret the data from the fsa or hid files Instruments Refer to I
149. ucts as set forth in the Life Technologies General Terms and Conditions of Sale found on Life Technologies website at www lifetechnologies com termsandconditions If you have any questions please contact Life Technologies at www lifetechnologies com support 132 AmpFtSTR MiniFiler PCR Amplification Kit User Guide Symbols sample files 34 A nucleotide addition defined 78 efficiency of 78 Numerics 310 instrument 31 3100 3100 Avant instruments 27 3130 3130xl instruments 27 3500 3500xL instruments 29 A accuracy and reproducibility 68 alleles off ladder 69 allelic dropout 103 allelic ladder profile 13 requirements for accurate genotyping 26 volume per reaction 28 30 32 amplification using bloodstained FTA cards 22 artifacts 79 baseline noise examples 80 bins check version 47 import 34 48 biohazard safety 125 buffer new 98 C characterization of loci validation 81 chemical safety 124 contents of kit 16 control DNA 9947A 17 control DNA about 17 cycle number validation 67 AmpFtSTR MiniFiler PCR Amplification Kit User Guide Index D data accuracy precision reproducibility 68 for different populations 90 93 Data Collection Software 15 degraded DNA 105 developmental validation 65 DNA degraded 105 effect of quantity figure 83 negative control reaction 21 positive control reaction 21 quantification 19 quantification methods 20 sample preparation 21 sensitivity 83 test sample
150. w document B March 2007 Add Experiments and Results chapter C December 2010 Change to limited licensing information D April 2011 Change to limited licensing information E March 2012 Change to limited licensing information F August 2012 Add 3500 and 3500xL Genetic Analyzer information Add D X Software information Add validation experiments and results for buffer and enzyme kit component changes Purpose The 5 MiniFiler PCR Amplification Kit User Guide provides information about the Life Technologies instruments chemistries and software associated with the AmpF STR MiniFiler PCR Amplification Kit AmpFtSTR MiniFiler PCR Amplification Kit User Guide 9 About This Guide Purpose 10 AmpFtSTR MiniFiler PCR Amplification Kit User Guide Overview B 11 Workflow overview 14 Instrument and software overview 15 Materials and equipment 16 Product overview Purpose Product description About the primers The AmpF STR MiniFiler PCR Amplification Kit is an assay optimized for genotyping degraded and or inhibited DNA samples It is a short tandem repeat STR multiplex assay optimized to allow direct amplification of single source sa
151. ware or Collection v1 2 or higher e Windows Vista Software v1 0 310 Windows XP 3 1 GeneMapper D 3 2 Windows NT and 3 0 GeneMapper D 3 2 Windows 2000 t We conducted validation studies for the MiniFiler Kit using these configurations Life Technologies 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 Multicomponent analysis is the process that separates the five different fluorescent dye colors into distinct spectral components The four dyes used in the MiniFiler Kit to label samples are 6 FAM VIC NED and PET dyes The fifth dye 1179 is used to label the GeneScan 500 LIZ Size Standard or the GeneScan 600 1129 Size Standard v2 0 Each of these fluorescent dyes emits its maximum fluorescence at a different wavelength During data collection on the Life Technologies 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 PET9 dye red and LIZ9 dye orange Although each of these dyes emits its maximum fluorescence at a different w
152. x the Master Mix thoroughly Primer Set exposed to too much light Store the Primer Set protected from light PCR System malfunction Refer to the thermal cycler user s manual and check instrument calibration Use of incorrect thermal cycling parameters Check the protocol for correct thermal cycling parameters MicroAmp Base used with tray retainer set and tubes in GeneAmp 9700 Remove MicroAmp Base from tray retainer set and repeat test Insufficient PCR product electrokinetically injected Prepare PCR product as described in Chapter 3 Perform Electrophoresis on page 25 Degraded formamide Check the storage of formamide do not thaw and refreeze multiple times Try Hi Di Formamide Positive signal from Control DNA 007 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 0 75 ng of DNA Repeat test Test sample contains high concentration of PCR inhibitor e g heme compounds certain dyes Quantitate DNA and add minimum necessary volume Repeat test Wash the sample in a Centricon 100 Repeat test Test sample DNA is severely degraded If possible evaluate the quality of DNA sample by running an agarose gel If DNA is degraded re amplify with an increased amount of DNA Dilution of test sample DNA in water or wrong buffer for example wrong EDTA concentration
153. ysis Methods SWGDAM July 10 2003 Based on these guidelines we 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 we performed and provides examples of results obtained We chose conditions that produced optimum 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 of STR kits intended for forensic and or parentage testing use IMPORTANT Each laboratory using the MiniFiler Kit must perform internal validation studies Developmental validation SWGDAM guideline 1 2 1 SWGDAM guideline 2 10 1 Developmental validation is the demonstration of the accuracy precision and reproducibility of a procedure by the manufacturer technical organization academic institution government laboratory or other party SWGDAM July 2003 The reaction conditions needed to provide the required degree of specificity and robustness must be determined These include thermocycling parameters the concentration of primers magnesium chloride DNA polymerase and other critical reagents SWGDAM July 2003 AmpFtSTR MiniFiler PCR Amplification Kit User Guide 65 g lt o e 3 D e 5 E s
154. ysis of degraded DNA J Forensic Sci 48 1054 1064 Chakraborty Kimmel M Stivers D Davison L and Deka 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 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 1988 A potent inhibitor of Taq DNA polymerase copurifies with human genomic DNA Nucleic Acids Res 16 10355 AmpFtSTR MiniFiler PCR Amplification Kit User Guide 127 Bibliography 128 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 commerc

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