A duplex real-time qPCR assay for the quantification of human nuclear and mitochondrial DNA in forensic samples: implications for quantifying DNA in degraded samples

A duplex real-time qPCR assay was developed for quantifying human nuclear and mitochondrial DNA in forensic samples. The nuclear portion of the assay utilized amplification of a approximately 170-190 bp target sequence that spans the repeat region of the TH01 STR locus, and the mitochondrial portion of the assay utilized amplification of a 69 bp target sequence in the ND1 region. Validation studies, performed on an ABI 7000 SDS instrument using TaqMan detection, demonstrated that both portions of the duplex assay provide suitable quantification sensitivity and precision down to 10-15 copies of each genome of interest and that neither portion shows cross-reactivity to commonly encountered non-human genomes. As part of the validation studies, a series of DNase-degraded samples were quantified using three different methods: the duplex nuclear-mitochondrial qPCR assay, the ABI Quantifiler Human DNA Quantification Kit qPCR assay, which amplifies and detects a 62 bp nuclear target sequence, and slot blot hybridization. For non-degraded and moderately degraded samples in the series, all three methods were suitably accurate for quantifying nuclear DNA to achieve successful STR amplifications to yield complete profiles using the ABI AmpFlSTR Identifiler kit. However, for highly degraded samples, the duplex qPCR assay provided better estimates of nuclear template for STR amplification than did either the commercial qPCR assay, which overestimated the quantity of STR-sized DNA fragments, leading to an increased proportion of undetected alleles at the larger STR loci, or slot blot hybridization, which underestimated the quantity of nuclear DNA, leading to an increased proportion of STR amplification artifacts due to amplification of excess template.