July 2022 ® Investigator Argus X-12 QS Handbook For multiplex amplification of 12 STR loci of the X- chromosome, plus Amelogenin and D21S11 Sample to Insight 1 [brand] [Name] Kit/Reagent Instructions for Use 07/2022 Contents Kit Contents ............................................................................................................... 4 Storage ..................................................................................................................... 5 Intended Use .............................................................................................................. 5 Safety Information ....................................................................................................... 6 Quality Control ........................................................................................................... 6 Introduction ................................................................................................................ 7 Equipment and Reagents to Be Supplied by User .......................................................... 11 Protocol: PCR Amplification ........................................................................................ 13 Protocol: Electrophoresis Using the ABI PRISM 310 Genetic Analyzer ............................. 16 Matrix generation ........................................................................................... 16 Sample preparation ........................................................................................ 21 Setting up the GeneScan Software ................................................................... 22 Analysis parameters ....................................................................................... 23 Protocol: Electrophoresis Using the ABI PRISM 3100-Avant/3100 Genetic Analyzer ........ 24 Spectral calibration/matrix generation .............................................................. 24 Sample preparation ........................................................................................ 29 Setting up the GeneScan Software ................................................................... 30 Analysis parameters ....................................................................................... 33 Protocol: Electrophoresis Using the Applied Biosystems 3130/3130xl Genetic Analyzer .. 34 Spectral calibration/matrix generation .............................................................. 34 Checking the matrix ........................................................................................ 40 2 Investigator Argus X-12 Instructions for Use (Handbook) 07/2022 Sample preparation ........................................................................................ 40 Setting up the Data Collection Software ............................................................ 41 Analysis parameters/analysis method ............................................................... 45 Protocol: Electrophoresis Using the Applied Biosystems 3500/3500xL Genetic Analyzer .. 46 Spectral calibration/matrix generation .............................................................. 47 Analysis parameters/analysis method ............................................................... 61 Protocol: Analysis ..................................................................................................... 62 Analysis software ........................................................................................... 62 Controls ........................................................................................................ 63 Alleles ........................................................................................................... 65 Quality Sensor ............................................................................................... 67 Characteristics and Use of X-chromosomal STRs ........................................................... 69 Calculation of population-genetic data .............................................................. 69 Variant alleles and mutations ..................................................................................... 73 Troubleshooting Guide .............................................................................................. 75 References ............................................................................................................... 79 Ordering Information ................................................................................................ 82 Kit Contents Investigator Argus X-12 QS Kit (100) (100) Catalog no. 383223 383225 Number of 25 µl reactions 25 100 Fast Reaction Mix 2.0* 750 µl 750 µl Primer Mix Argus X-12 QS 63 µl 250 µl Nuclease-free water 1.9 ml 1.9 ml Control DNA 9947A 200 µl 200 µl DNA size standard 550 (BTO) 13 µl 50 µl Allelic ladder Argus X-12 QS 25 µl 25 µl Quick Start Protocol 1 1 * C ontains DNA Polymerase, dNTPs, MgCl2 and bovine serum albumin (BSA) 4 Investigator Argus X-12 Instructions for Use (Handbook) 07/2022 Storage The Investigator Argus X-12 QS Kit is shipped on dry ice. It should be stored immediately upon receipt at –30°C to –15°C in a constant-temperature freezer. Avoid repeated thawing and freezing. The Primer Mix and allelic ladder must be stored protected from the light. DNA samples and post-PCR reagents (allelic ladder and DNA size standard) should be stored separately from the PCR reagents. Under these conditions, the components are stable until the expiration date indicated on the kit. Once opened, the Investigator Argus X-12 QS Kit can be stored at 2–8°C for a maximum of 3 months or re-frozen and stored at –30°C to –15°C in a constant-temperature freezer for longer periods. Intended Use The Investigator Argus X-12 QS Kit is intended for molecular biology applications in forensic, human identity, and paternity testing. This product is not intended for the diagnosis, prevention, or treatment of a disease. All due care and attention should be exercised in the handling of the products. We recommend all users of QIAGEN® products to adhere to the NIH guidelines that have been developed for recombinant DNA experiments, or to other applicable guidelines. Safety Information When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective goggles. For more information, please consult the appropriate safety data sheets (SDSs). These are available online in convenient and compact PDF format at www.qiagen.com/safety where you can find, view, and print the SDS for each QIAGEN kit and kit component. CAUTION: DO NOT add bleach or acidic solutions directly to the sample preparation waste. Quality Control In accordance with QIAGEN’s ISO-certified Quality Management System, each lot of Investigator Argus X-12 QS Kit is tested against predetermined specifications to ensure consistent product quality. Investigator Argus X-12 QS Kits meet ISO 18385 requirements. 6 Investigator Argus X-12 Instructions for Use (Handbook) 07/2022 Introduction The Investigator Argus X-12 QS Kit is a multiplex application for 12 X-chromosomal short tandem repeat (STR) loci. The kit supplements the Investigator Argus Y- 12 QS Kit for kinship and paternity testing, especially in complicated deficiency cases. The Investigator Argus X-12 QS Kit contains primers for Amelogenin (AM) for gender determination, DXS7132, DXS7423, DXS8378, DXS10074, DXS10079, DXS10101, DXS10103, DXS10134, DXS10135, DXS10146, DXS10148 and HPRTB. The markers are clustered into 4 linkage groups (3 markers per group; Table 1), and thus each set of 3 markers is handled as a haplotype for genotyping (Figure 1, next page). Furthermore, the autosomal STR marker D21S11 is included as a consistent marker for common database kits and prevents sample mixup. The Investigator Argus X-12 QS Kit Primer Mix contains an internal PCR control (Quality Sensor QS1) to provide helpful information about the efficiency of the PCR. The Quality Sensor is amplified simultaneously with the polymorphic STR markers. The Investigator Argus X-12 QS Kit is specifically for rapid and reliable generation of DNA profiles from blood, buccal swabs and forensic stains. The kit utilizes QIAGEN’s fast-cycling PCR technology, allowing amplification in around 80 minutes. It provides highly robust results with inhibitor-resistant chemistry. The primers are fluorescence-labeled with the following dyes:  6-FAM™: QS1, Amelogenin, DXS10103, DXS8378, DXS10101, DXS10134  BTG: DXS10074, DXS7132, DXS10135  BTY: DXS7423, DXS10146, DXS10079  BTR: HPRTB, DXS10148, D21S11 Table 1. Linkage groups Linkage group 1 (Xp22) DXS8378 – DXS10135 – DXS10148 2 (Xp11) DXS7132 – DXS10074 – DXS10079 3 (Xp26) HPRTB – DXS10101 – DXS10103 4 (Xp28) DXS7423 – DXS10134 – DXS10146 The optimal amount of DNA under standard conditions is 0.2–0.5 ng. Internal validations demonstrated reliable results with <0.1 ng DNA. The Investigator Argus X-12 QS Kit was validated using the GeneAmp® PCR System 9700 (with Gold-plated 96-Well Silver Block) and the Applied Biosystems® 3500™ Genetic Analyzer. The Forensic ChrX Research Group initiated the online data base ChrX-STR.org (http://www.chrx-str.org) that calculates population-genetic data on basis of X-chromosomal allele frequencies (1). 8 Investigator Argus X-12 Instructions for Use (Handbook) 07/2022 Amelogenin X Linkage group 1 DXS10148 9 Mb DXS10135 DXS8378 DXS7132 66 Mb DXS10079 DXS10074 Linkage group 2 Linkage group 3 133 Mb DXS10103 HPRTB DXS10101 149 Mb DXS10146 DXS10134 Linkage group 4 DXS7423 Figure 1. The ideogram of the X-chromosome describes the physical localization of the STR loci that can be analyzed using the Investigator Argus X-12 QS Kit. Distances from the p-telomere are shown in Mb (www.ncbi.nlm.nih.gov/genome/guide/human as of 11/2014). Table 2 and Table 3 show the STR loci with their chromosomal mapping, repeat motifs and alleles that are concordant with the International Society for Forensic Genetics (ISFG) guidelines for the use of microsatellite markers (2). Table 2. Locus-specific information of the Investigator Argus X-12 QS Kit Locus GenBank® Accession number Repeat motif of the reference allele Chromosomal mapping Amelogenin X M55418 – Xp22.1-22.3 Amelogenin Y M55419 – Yp11.2 DXS7132 G08111 [TCTA]13 Xq11.2 DXS7423 AC109994 [TCCA]3 TCTGTCCT [TCCA]12 Xq28 DXS8378 G08098 [CTAT]12 Xp22.31 DXS10074 AL356358 [AAGA]14 Xq12 DXS10079 AL049564 [AGAG]3 TGAAAGAG [AGAA]17 Xq12 AGAG [AGAA]3 AC004383 [AAAG]3 GAAAGAAG [GAAA]3 A DXS10101 [GAAA]4 AAGA [AAAG]5 Xq26.2 AAAAAGAA [AAAG]13 AA DXS10103 BV680555 [TAGA]2 CTGA Xq26.2 [CAGA][TAGA]11[CAGA]4[TAGA] [GAAA]3 GAGA [GAAA]4 AA DXS10134 AL034384 [GAAA] GAGA [GAAA]4 GAGA Xq28 [GACAGA]3 [GAAA] GTAA [GAAA]3 AAA [GAAA]4 AAA [GAAA]15 DXS10135 AC003684 [AAGA]3 GAAAG [GAAA]20 Xp22.31 [TTCC]3 T [TTCC]3 TTTC CTCCCTTCC DXS10146 AL034384 [TTCC] [TCCC] TTCTTCTTTC [TTCC]2 Xq28 TTTCTT [CTTT]2 CTTC [CTTT]10 T [CTTT]2 DXS10148 AC003684 [GGAA]4[AAGA]12[AAAG]4 N8 Xp22.31 [AAGG]2 HPRTB M26434 [AGAT]12* Xq26.2 D21S11 AP000433 [TCTA]4 [TCTG]6 [TCTA]3 TA [TCTA]3 21q21.1 TCA [TCTA]2 TCCATA [TCTA]11 * [AGAT] is the most common repeat structure; for variations see the NIST and reference 3. 10 Investigator Argus X-12 Instructions for Use (Handbook) 07/2022