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
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.
Available from: Samantha A Schrier Vergano
- "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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ABSTRACT: Progressive multisystem disease should invoke consideration of potential mitochondrial etiologies. Mitochondrial disease can affect any organ system at any time, particularly involving neurologic, cardiac, muscular, gastroenterologic, and/or ophthalmologic manifestations. We report here a 19-year-old Caucasian man who was followed since birth in multiple pediatric subspecialty clinics for myelomeningocele complications. However, he progressively developed a host of additional problems that were not readily attributable to his neural tube defect involving developmental, ophthalmologic, cardiac, muscular, endocrine, and intermediary metabolic manifestations. Clinical diagnostic testing limited to analysis for common point mutations and deletions in his blood mitochondrial DNA (mtDNA) was not revealing. Skeletal muscle biopsy revealed abnormal mitochondrial morphology and immunostaining, mitochondrial proliferation, and mildly reduced respiratory chain complex I-III activity. Whole mitochondrial genome sequencing analysis in muscle identified an apparently homoplasmic, novel, m.12264C>T transition in the tRNA serine (AGY) gene. The pathogenicity of this mutation was supported by identification of it being present at low heteroplasmy load in his blood (34%) as well as in blood from his maternal grandmother (1%). The proband developed severe nuclear cataracts that proved to be homoplasmic for the pathogenic mtDNA m.12264C>T mutation. This case highlights the value of pursuing whole mitochondrial genome sequencing in symptomatic tissues in the diagnostic evaluation of suspected mitochondrial disease. Furthermore, it is the first report to directly implicate a single mtDNA mutation in the pathogenesis of ocular cataracts and clearly illustrates the important contribution of normal metabolic activity to the function of the ocular lens.
Available from: Martin Horlitz
- "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 . "
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ABSTRACT: Duplex real-time PCR assays have been widely used to determine amounts and concentrations of free circulating DNA in human blood plasma samples. Circulatory plasma DNA is highly fragmented and hence a PCR-based determination of DNA concentration may be affected by the limited availability of full-length targets in the DNA sample. This leads to inaccuracies when counting PCR target copy numbers as whole genome equivalents.
A model system was designed allowing for assessment of bias in a duplex real-time PCR research assay. We collected blood plasma samples from male donors in pools of 6 to 8 individuals. Circulatory plasma DNA was extracted and separated by agarose gel electrophoresis. Separated DNA was recovered from the gel in discrete size fractions and analyzed with different duplex real-time PCR Taqman assays detecting a Y chromosome-specific target and an autosomal target. The real-time PCR research assays used differed significantly in their ability to determine the correct copy number ratio of 0.5 between Y chromosome and autosome targets in DNA of male origin. Longer PCR targets did not amplify quantitatively in circulatory DNA, due to limited presence of full-length target sequence in the sample.
PCR targets of the same small size are preferred over longer targets when comparing fractional circulatory DNA concentrations by real-time PCR. As an example, a DYS14/18S duplex real-time PCR research assay is presented that correctly measures the fractional concentration of male DNA in a male/female mixture of circulatory, fragmented DNA.
Available from: Thorsten Schwark
- "Various methods (e.g. real time PCR) to detect PCR inhibitors  or detect human specific DNA fragments   have been utilized to optimize analysis of problematic forensic trace material. "
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ABSTRACT: The analysis of short tandem repeats is one of the most powerful tools in forensic genetics. Forensic practice sometimes requires the individualization of samples that may contain only highly degraded nuclear DNA, mitochondrial DNA or PCR inhibitors that hamper DNA amplification. We designed a new multiplex PCR with reduced size amplicons (<200 bp), providing a double sex determination (amelogenin plus two Y-STRs), the detection of two autosomal markers and the amplification of mitochondrial specific fragments from the hypervariable region I (HVI). Additionally, a quality sensor was developed to check for the presence of any PCR inhibitors. The new multiplex PCR shows a reproducible detection threshold down to 25 pg and gives signals even out of highly degraded materials. All signals are reproducible and reliable as it could be shown in comparison to results from commercially available STR multiplex-PCRs. In no case DNA fragments were detectable using any other assay when the quality sensor was not detectable. There was a good correlation between detection of mitochondrial specific fragments in the multiplex-PCR and success of subsequent sequencing of HVI region. The same could be shown for STR analysis: Most samples successfully analyzed in our PCR yielded at least a partial STR profile using a commercial STR kit. We present an assay that allows an easy, reliable, and cost efficient evaluation of DNA sample quality combined with a first rough sample individualization and sex determination suitable for forensic purposes. This assay may help the forensic lab personnel to decide on further sample processing.
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