This study examined the forensic utility of allele-discriminatory quantitative real-time PCR (nPCR) using Minor Groove Binder TaqMan probes, targeting the highly variable mitochondrial single nucleotide polymorphism 16519T/C.
The performance of an allele-discriminatory quantitative TaqMan nPCR SNP assay was assessed with respect to parameters relevant to mtDNA forensic typing: sensitivity, specificity, range of suitable template concentrations, mixture/heteroplasmy detection, reproducibility, ease of data analysis, and quantitative performance. In all these areas, the assay performed well for forensic purposes Capabilities were particularly strong for quantifying allele proportions, contamination avoidance due to homogeneous assay format, and absolute template copy number quantitation. The quantitative nature of the TaqMan assay makes it unique among recent SNP typing methods. It is potentially useful for distinguishing between authentic signal and possible contamination based on measurable values. These features make it a useful tool for forensic applications, complementing endpoint-signal-based, multi-step SNP typing approaches with a high multiplexing capability, such as single nucleotide primer extension assays. A total of 405 genomic DNA (gDNA) samples from healthy west Eurasian donors from Austria were classified as either homoplasmic (n = 403) or heteroplasmic (n =2) at the mitochondrial nucleotide position (ntp) 16519 on the basis of sequence information from a previous study. The sample set included the heteroplasmic reference samples B and C and the homoplasmic samples A (16519T) and D (16519C). In addition to describing the DNA samples, this article explains the design of primers and hybridization probes; dual probe singleplex nPCR; the preparation of plasmid DNA standards; PCR efficiency, linear dynamic range, and absolute quantification; allelic discrimination and allele proportions in mixed/heteroplasmic samples; and statistical analyses. 2 figures, 1 table, and 10 references
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