Amplification Kits GlobalFiler and GlobalFiler IQC PCR

1 For Research, Forensic, or Paternity Use Only. Not for use in diagnostic and GlobalFiler IQC PCRA mplification KitsUSER GUIDEfor use with: GlobalFiler PCR Amplification Kit (Cat. No. 4476135 and 4482815) GlobalFiler IQC PCR Amplification Kit (Cat. No. A43565)Publication Number 4477604 Revision FLife Technologies Ltd | 7 Kingsland Grange | Woolston, Warrington WA1 4SR | United KingdomFor descriptions of symbols on product labels or product documents, go to information in this guide is subject to change without : TO THE EXTENT ALLOWED BY LAW, THERMO FISHER SCIENTIFIC INC. AND/OR ITS AFFILIATE(S) WILL NOT BE LIABLE FOR SPECIAL,INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT,INCLUDING YOUR USE OF history: Pub. No. 4477604 RevisionDateDescriptionF03 October 2019 Add information for the GlobalFiler IQC PCR Amplification Kit: pages 9 12, 14, 16,18 19, 26, 30, 36 40, 43 45, 50, 52 56, 149 170, 3500 software information: pages 19, 28, 175, GeneMapper ID X Software: pages 176, July 2016 Add information on 30 cycles with 500-ng sample concentration to Chapter 1,Chapter 2, and Chapter 5.

2 Add references to 3500 Series Data Collection 3 andGeneMapper ID-X Add section on Amplification artifacts to Chapter changes: Reorganize Chapter 1 and Chapter 2015 Remove Master Mix Additive. Add the ProFlex PCR Systems. Add information aboutartifacts identified at the TH01 locus and TPOX locus. Add Section Performanceverification after BSA component replacement . Update 2014 Change storage conditions from 2 months to 6 months. Add Veriti Thermal Cyclerverification 2014 Add data to Chapter 5 concerning the evaluation of Hardy-Weinberg equilibrium. Addinformation about the Quantifiler HP and Trio DNA Quantification kits . Update thetroubleshooting 2013 New documentTrademarks: All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. Windows is a trademarks ofMicrosoft Corporation. FTA is a trademark of GE Healthcare. NUCLEIC-CARD is a trademark of Copan Italia , and used by Thermo FisherScientific under their permission.

3 MiniTab is a trademark of Minitab JMP is a trademark of SAS Institute 2019 Thermo Fisher Scientific Inc. All rights CHAPTER 1 Product DNA input amounts and PCR cycles ..10 About the used in the amplified by the and controls that are Allelic Ladder IQC Allelic Ladder kit DNA Control 007 IQC kit DNA Control 007 and kit contents and IQC kit contents and materials not and software CHAPTER 2 Perform PCR ..21 DNA of quantification before STR of DNA quantity on of quantifying you low-TE reagents (before first use of the kit)..23 Prepare the Amplification kit Amplification ( GlobalFiler kit only)..26 GlobalFiler and GlobalFiler IQC PCR Amplification kits user Guide3 CHAPTER 3 Perform ladder requirements for required for up the 3500/3500xL instruments for electrophoresis (before first use of the kit)..28 Electrophoresis software spectral up the 3130/3130xl instruments for electrophoresis (before first use of the kit).

4 30 Electrophoresis software and activate 6-dye spectral samples for electrophoresis (3500 Series and 3130 Series instruments)..32 CHAPTER 4 Analyze data with GeneMapper ID X Software ..34 Overview of GeneMapper ID X ladder requirements for data names and versions used in this up the GeneMapper ID X Software for analysis (before first use of the kit)..36 Workflow: Set up GeneMapper ID X Software ..36 Check panel, bin, and stutter file versions on your (If needed) Download newer versions of panel, bin, and stutter files ..37 Import panels, bins, and marker (Optional) Define custom table or plot an analysis an analysis method a size standard definition file if the GS600_LIZ_ (60 460) size standard definition file ..48 Create a size standard definition and edit sample files with GeneMapper ID X Software ..50 Examine or edit a more information on using the GeneMapper ID X Software ..51 CHAPTER 5 Assess the PCR reaction with the InternalQuality Control of the Internal Quality Control the PCR slope profile with decreased or missing IQCL peak height (inhibition).

5 54 Ski slope profile with balanced IQC peaks (degradation)..55No sample peaks with balanced IQC sample peaks with extreme inhibition of IQC peaks ..56 Contents4 GlobalFiler and GlobalFiler IQC PCR Amplification kits user guide CHAPTER 6 Experiments and of requirements for internal guideline guideline cycling cycle , precision, and guideline and size window peaks in the of extra peaks: peaks: Addition of 3' A peaks: peaks: Amplification -related of guideline in this of polymorphisms .. Guideline study guideline of DNA quantity on guideline of inhibitors of inhibitors humic guideline study study and GlobalFiler IQC PCR Amplification kits user Guide5 Resolution of genotypes in mixed of detection of the minor guideline data in the samples used in these of Identity of identity of paternity exclusion CHAPTER 7 Performance verification after BSAcomponent component and study study results: Allele recovery, average peak height, stuttercomparison, and study results: Intralocus, intracolor, and intercolor study study study CHAPTER 8 Performance verification with 30 PCR cycles.

6 142 Experiment , methods, and samples ..143 Minimum threshold and contamination study study sample study sample and GlobalFiler IQC PCR Amplification kits user guide CHAPTER 9 Performance verification with the GlobalFiler IQC , methods, and samples ..150 Materials .. and test cases ..150 Sensitivity study sample study results ..153 Degraded sample study results ..155 PCR component concentration analysis cycle number analysis peak sizing specificity concordance with population samples and NIST study peaks in the peaks: peaks: IQCS artifact peaks ..168 Results summary .. APPENDIX A APPENDIX B Materials required but not kit preparation required cycler required PCR Thermal Cycler Thermal PCR System analyzer required Series Genetic Series Genetic software required ID X required and and GlobalFiler IQC PCR Amplification kits user Guide7 APPENDIX C PCR work area setup and lab setup work area DNA work APPENDIX D hazard Documentation and and technical product and GlobalFiler IQC PCR Amplification kits user GuideProduct information Product Contents and Required materials not Instrument and software !

7 Before using this product, read and understand the information in the Safety appendix in this descriptionThe Applied Biosystems GlobalFiler and GlobalFiler IQC PCR Amplification Kitsare 6-dye, short tandem repeat (STR) multiplex assays for the Amplification of humangenomic kits amplify: 21 autosomal STR loci (D3S1358, vWA, D16S539, CSF1PO, TPOX, D8S1179,D21S11, D18S51, D2S441, D19S433, TH01, FGA, D22S1045, D5S818, D13S317,D7S820, SE33, D10S1248, D1S1656, D12S391, D2S1338) 1 Y-STR (DYS391) 1 insertion/deletion polymorphic marker on the Y chromosome (Y indel) Amelogenin (sex determining marker)Both kits contain the 13 original CODIS loci with 7 from the expanded EuropeanStandard Set of Loci (ESSL) and the highly discriminating SE33 locus. The kits delivera 24-locus multiplex, high discrimination power, high sensitivity, and tolerance toinhibitors. The concentration of 10 mini-STR loci that are entirely below 220 bpmaximizes performance on degraded samples.

8 The highly optimized bufferformulation contains an enzyme that allows completion of Amplification in~80 addition to the loci listed above, the GlobalFiler IQC PCR Amplification Kitamplifies 2 Internal Quality Control (IQC) more information on the IQC markers, see Chapter 5, Assess the PCR reactionwith the Internal Quality Control System .Note: The primer set and the allelic ladder in the GlobalFiler kit are different thanthe primer set and the allelic ladder in the GlobalFiler IQC kit. Do not overviewGlobalFiler and GlobalFiler IQC PCR Amplification kits user Guide9 The kits are validated for use with: ng DNA (15 L input volume) for 29 cycles 500 pg DNA (15 L input volume) for 30 cycles for increased sensitivity withlow-concentration samplesThe GlobalFiler and GlobalFiler IQC kit primers are manufactured using the samesynthesis and purification improvements as the primers in the NGM SElect and theIdentifiler Plus kits .

9 These improvements enhance the assay signal-to-noise ratio andsimplify the interpretation of primers used in the kits are: For all loci except AMEL The same primer sequences as the NGM SElect kitand the Identifiler Plus kit including SNP-specific primers for the vWA,D16S539, AMEL, D2S441, D22S1045, and D8S1179 loci. For AMEL The same primer sequences as the NGM SElect kit (which aredifferent from the Identifiler Plus kit).The kits also include the following primer additions and modifications: Addition of DYS391 and a novel Y indel. The TPOX reverse primer has been redesigned to relocate the amplicon into thehigher size range of the multiplex and optimize marker spacing. Addition of 8 new SNP-specific primers for the D3S1358, vWA, D18S51, D19S433,TH01, FGA, D5S818, and SE33 loci. The second degenerate primer was added tothe vWA locus to address two different SNPs in the primer binding GlobalFiler IQC kit contains additional PCR primers, which are the same IQCprimers contained in the NGM Detect PCR Amplification Kit.

10 The IQCS (small) andIQCL (large) markers are amplified from a synthetic, plasmid-borne DNA templatethat is present in low copy number in the GlobalFiler IQC Primer Mix. The primersamplify the Internal Quality Control ("IQC") markers in the blue FAM dye more information, see Chapter 5, Assess the PCR reaction with the InternalQuality Control System .Non-nucleotide linkers are used in primer synthesis for the following loci: D19S433,vWA, CSF1PO, D2S441, TH01, FGA, and D12S391. For these primers, non-nucleotidelinkers are placed between the primers and the fluorescent dye during oligonucleotidesynthesis (Butler 2005, Grossman et al., 1994). Non-nucleotide linkers enablereproducible positioning of the alleles to facilitate interlocus spacing. The combinationof a 6-dye fluorescent system and the use of non-nucleotide linkers allowssimultaneous Amplification and efficient separation of all 24 markers duringautomated DNA fragment FAM BlueSamples, allelic ladders, and controlsVIC GreenNED YellowTAZ RedValidated DNAinput amounts andPCR cyclesAbout the primersDyes used in thekitChapter 1 Product informationProduct description110 GlobalFiler and GlobalFiler IQC PCR Amplification kits user GuideDyeColorLabelSID PurpleSamples, allelic ladders, and controlsLIZ OrangeGeneScan 600 LIZ Size Standard.