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Development and validation of InnoQuant™, a sensitive human DNA quantitation and degradation assessment method for forensic samples using high copy number mobile elements Alu and SVA

      Highlights

      • A qPCR assay is described which provides an assessment of sample quality.
      • The multiplex is a TaqMan assay comprised of three fluorescently labeled probes.
      • Three targets are studied: autosomal 80 bp, autosomal 207 bp, and a synthetic IPC.
      • Developmental validation studies are described.
      • InnoQuant™ proves to be a useful tool for forensic DNA testing laboratories.

      Abstract

      There is a constant need in forensic casework laboratories for an improved way to increase the first-pass success rate of forensic samples. The recent advances in mini STR analysis, SNP, and Alu marker systems have now made it possible to analyze highly compromised samples, yet few tools are available that can simultaneously provide an assessment of quantity, inhibition, and degradation in a sample prior to genotyping. Currently there are several different approaches used for fluorescence-based quantification assays which provide a measure of quantity and inhibition. However, a system which can also assess the extent of degradation in a forensic sample will be a useful tool for DNA analysts. Possessing this information prior to genotyping will allow an analyst to more informatively make downstream decisions for the successful typing of a forensic sample without unnecessarily consuming DNA extract. Real-time PCR provides a reliable method for determining the amount and quality of amplifiable DNA in a biological sample.
      Alu are Short Interspersed Elements (SINE), approximately 300 bp insertions which are distributed throughout the human genome in large copy number. The use of an internal primer to amplify a segment of an Alu element allows for human specificity as well as high sensitivity when compared to a single copy target. The advantage of an Alu system is the presence of a large number (>1000) of fixed insertions in every human genome, which minimizes the individual specific variation possible when using a multi-copy target quantification system. This study utilizes two independent retrotransposon genomic targets to obtain quantification of an 80 bp “short” DNA fragment and a 207 bp “long” DNA fragment in a degraded DNA sample in the multiplex system InnoQuant™. The ratio of the two quantitation values provides a “Degradation Index”, or a qualitative measure of a sample's extent of degradation. The Degradation Index was found to be predictive of the observed loss of STR markers and alleles as degradation increases. Use of a synthetic target as an internal positive control (IPC) provides an additional assessment for the presence of PCR inhibitors in the test sample.
      In conclusion, a DNA based qualitative/quantitative/inhibition assessment system that accurately predicts the status of a biological sample, will be a valuable tool for deciding which DNA test kit to utilize and how much target DNA to use, when processing compromised forensic samples for DNA testing.

      Keywords

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      References

        • Barbisin M.
        • Fang R.
        • O'Shea C.E.
        • Calandro L.M.
        • Furtado M.R.
        • Shewale J.G.
        Developmental validation of the Quantifiler Duo DNA Quantification kit for simultaneous quantification of total human and human male DNA and detection of PCR inhibitors in biological samples.
        J. Forensic Sci. 2009; 54: 305-319
        • Krenke B.E.
        • Nassif N.
        • Sprecher C.J.
        • Knox C.
        • Schwandt M.
        • Storts D.R.
        Developmental validation of a real-time PCR assay for the simultaneous quantification of total human and male DNA.
        Forensic Sci. Int.: Genet. 2008; 3: 14-21
        • Qiagen
        Investigator® Quantiplex HYres Kit Validation Report 11/2012.
        2012 (Available from: http://www.qiagen.com/products/catalog/assay-technologies/investigator-quantiplex-hyres-kit#resources)
        • Shewale J.G.
        • Schneida E.
        • Wilson J.
        • Walker J.A.
        • Batzer M.A.
        • Sinha S.K.
        Human genomic DNA quantitation system, H-Quant: development and validation for use in forensic casework.
        J. Forensic Sci. 2007; 52: 364-370
        • Wang H.
        • Xing J.
        • Grover D.
        • Hedges D.J.
        • Han K.
        • Walker J.A.
        • Batzer M.A.
        SVA elements: a hominid-specific retroposon family.
        J. Mol. Biol. 2005; 354: 994-1007
        • Nicklas J.A.
        • Noreault-Conti T.
        • Buel E.
        Development of a real-time method to detect DNA degradation in forensic samples.
        J. Forensic Sci. 2012; 57: 466-471
        • Swango K.L.
        • Hudlow W.R.
        • Timken M.D.
        • Buoncristiani M.R.
        Developmental validation of a multiplex qPCR assay for assessing the quantity and quality of nuclear DNA in forensic samples.
        Forensic Sci. Int. 2007; 170: 35-45
        • Walker J.A.
        • Hedges D.J.
        • Perodeau B.P.
        • Landry K.E.
        • Stoilova N.
        • Laborde M.E.
        • Shewale J.
        • Sinha S.K.
        • Batzer M.A.
        Multiplex polymerase chain reaction for simultaneous quantitation of human nuclear, mitochondrial, and male Y-chromosome DNA: application in human identification.
        Anal. Biochem. 2005; 337: 89-97
        • Walker J.A.
        • Kilroy G.E.
        • Xing J.
        • Shewale J.
        • Sinha S.K.
        • Batzer M.A.
        Human DNA quantitation using Alu element-based polymerase chain reaction.
        Anal. Biochem. 2003; 315: 122-128
        • Batzer M.A.
        • Deininger P.L.
        Alu repeats and human genomic diversity.
        Nat. Rev. Genet. 2002; 3: 370-379
        • Carter A.B.
        • Salem A.H.
        • Hedges D.J.
        • Keegan C.N.
        • Kimball B.
        • Walker J.A.
        • Watkins W.S.
        • Jorde L.B.
        • Batzer M.A.
        Genome-wide analysis of the human Alu Yb-lineage.
        Hum. Genomics. 2004; 1: 167-178
        • Shewale J.G.
        • Liu R.H.
        Forensic DNA Analysis: Current Practices and Emerging Technologies.
        CRC Press: Taylor & Francis Group, Boca Raton, FL, United States2014 (405pp.)
        • Nicklas J.A.
        • Buel E.
        Development of an Alu-based, real-time PCR method for quantitation of human DNA in forensic samples.
        J. Forensic Sci. 2003; 48: 936-944
        • Nicklas J.A.
        • Buel E.
        Simultaneous determination of total human and male DNA using a duplex real-time PCR assay.
        J. Forensic Sci. 2006; 51: 1005-1015
        • LaSalle H.E.
        • Duncan G.
        • McCord B.
        An analysis of single and multi-copy methods for DNA quantitation by real-time polymerase chain reaction.
        Forensic Sci. Int. Genet. 2011; 5: 185-193
        • Damert A.
        • Raiz J.
        • Horn A.V.
        • Lower J.
        • Wang H.
        • Xing J.
        • Batzer M.A.
        • Löwer R.
        • Schumann G.G.
        5′-Transducing SVA retrotransposon groups spread efficiently throughout the human genome.
        Genome Res. 2009; 19: 1992-2008
      1. ChargeSwitch® gDNA Buccal Cell Kits For purification of genomic DNA from human buccal swabs. Invitrogen by Life Technologies User Guide Revision date 12 September 2012.

        • Budowle B.
        • Smith J.
        • Moretti T.
        • DiZinno J.
        DNA Typing Protocols: Molecular Biology and Forensic Analysis.
        Eaton Publishing, Natick2000: 41-42
        • Vallone P.M.
        • Kline M.C.
        • Duewer D.L.
        • Decker A.E.
        • Redman J.W.
        • Travis J.C.
        • Smith M.V.
        • Butler J.M.
        Development and usage of a NIST standard reference material for real time PCR quantitation of human DNA.
        Forensic Sci. Int. Genet. Suppl. Ser. 2008; 1: 80-82
        • Sinha S.K.
        • Montgomery A.H.
        • Pineda G.M.
        • Thompson R.
        • Klaskala L.
        Development of a highly sensitive quantitation system for assessing DNA Quality in forensic samples.
        Proc. Am. Acad. Forensic Sci. A. 2013; 173: 120
        • Konkel M.K.
        • Walker J.A.
        • Batzer M.A.
        LINEs and SINEs of primate evolution.
        Evolut. Anthropol. 2010; 19: 236-249
      2. Sinha S., Method for Genetic Detection using Interspersed Genetic Elements: a Multiplexed DNA Analysis System and Development of a Highly Sensitive Quantification System for Assessing DNA Quality in Forensic Samples. United States Patent Application Publication #20140051075A1, published on 20.02.14.

        • Untergrasser A.
        • Cutcutache I.
        • Koressaar T.
        • Ye J.
        • Faircloth B.C.
        • Remm M.
        • Rozen S.G.
        Primer3 – new capabilities and interfaces.
        Nucleic Acids Res. 2012; 40: e115
        • Koressaar T.
        • Remm M.
        Enhancements and modifications of primer design program Primer3.
        Bioinformatics. 2007; 23: 1289-1291
        • Wang D.Y.
        • Chang C.W.
        • Lagace R.E.
        • Calandro L.M.
        • Hennessy L.K.
        Developmental validation of the AmpFlSTR(R) Identifiler® Plus PCR Amplification Kit: an established multiplex assay with improved performance.
        J. Forensic Sci. 2012; 57: 453-465
        • Mulero J.J.
        • Chang C.W.
        • Lagacé R.E.
        • Wang D.Y.
        • Bas J.L.
        • McMahon T.P.
        • Hennessy L.K.
        Development and validation of the AmpFlSTR MiniFiler PCR Amplification Kit: a MiniSTR multiplex for the analysis of degraded and/or PCR inhibited DNA.
        J. Forensic Sci. 2008; 53: 838-852
      3. M.G. Ensenberger, P.M. Fulmer, The PowerPlex® 16 HS System, Profiles in DNA 12 [2009] 9–11, Promega Corporation. Available at https://www.promega.com/∼/media/files/resources/profiles%20in%20dna/1201/the%20powerplex%2016%20hs%20system.pdf?la=en.

        • Urban C.
        • Gruber F.
        • Kundi M.
        • Falkner F.G.
        • Dorner F.
        • Hammerle T.
        A systematic and quantitative analysis of PCR template contamination.
        J. Forensic Sci. 2000; 45: 1307-1311