Simultaneous Determination of Total Human and Male DNA Using a Duplex Real-Time PCR Assay

Vermont Forensic Laboratory, Department of Public Safety, 103 S. Main St., Waterbury, VT 05671, USA.
Journal of Forensic Sciences (Impact Factor: 1.16). 10/2006; 51(5):1005-15. DOI: 10.1111/j.1556-4029.2006.00211.x
Source: PubMed


A single duplex assay to determine both the amount of total human DNA and the amount of male DNA in a forensic sample has been developed. This assay is based on TaqMan technology and uses the multicopy Alu sequence to quantitate total human DNA and the multicopy DYZ5 sequence to quantitate Y chromosomal (male) DNA. The assay accepts a wide concentration range of input DNA (2 muL of 64 ng/microL to 0.5 pg/microL), and also allows detection of PCR failure. The PCR product sizes Alu (127 bp) and DYZ5 (137bp) approximate that of the smaller short tandem repeats (STRs) which should make the assay predictive of STR success with degraded DNA. The assay was optimized for probe/primer concentrations and BSA addition and validated on its reproducibility, on its human specificity, on its nonethnic variability, for artificial mixtures and adjudicated casework, for the effect of inhibitors and for state of DNA degradation. This assay should prove very usual in forensic analyses because knowing the relative amounts of male versus female DNA can allow the examiner to decide which samples may yield the most probative value in a case or direct the samples to methods that would yield the greatest information.

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    • "TaqMan qPCR analysis was performed on an ABI PRISM 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA). Relative expression was calculated as previously described (Ji et al., 2003; Nicklas & Buel, 2006). "
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    • "Correct, non-biased quantification of fragmented DNA is also important in forensic analysis of human DNA. An example is the determination of male DNA content of a case work DNA sample where male and total human DNA are quantitatively detected by a duplex real-time PCR assay in order to determine the percentage of male DNA in the sample [5]. "
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    • "Various methods (e.g. real time PCR) to detect PCR inhibitors [27] or detect human specific DNA fragments [28] [29] have been utilized to optimize analysis of problematic forensic trace material. "
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