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

Abstract

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 ∼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 R 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 nuclearmitochondrial qPCR assay, the ABI Quantifiler TM 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 R Identifiler TM 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.

A quantitative PCR assay for the assessment of DNA degradation in forensic samples

“…This approach has been used by Alonso et al [6] who have developed two singleplex qPCR assays to separately quantify short and long mitochondrial target sequences to assess degradation in forensic samples. As part of our previous development and validation of a duplex qPCR assay for quantifying nuclear and mitochondrial DNA [7], we have also shown that for highly degraded samples the quantity of DNA depends significantly on the length of the target sequence that is amplified in the assay. For highly fragmented DNA samples, a qPCR assay that amplified a 62 bp target sequence detected more DNA than a qPCR assay that amplified a $170-190 bp target, a result consistent with the allele-length-dependent STR intensity profiles that are commonly seen for degraded DNA [8].…”

Low volume amplification and sequencing of mitochondrial DNA on a chemically structured chip

“…To confirm the nuclear DNA content, all diluted samples were quantified again. To determine the corresponding mtDNA content, 800-900 mtDNA genome equivalents per cell [2,9,28], or rather 128±11 mtDNA genome equivalents per pg chromosomal DNA were calculated (personal communication: H. Niederstätter, GMI, Innsbruck, Austria).…”

Comparison of different protocols for DNA preparation and PCR amplification of mitochondrial genes of tardigrades

“…Thus, the specimens pass through a chemical and physical ageing process which also causes a reduced survival rate in tardigrades after rehydration . For degraded template DNAs other pairs of primers for small amplicons or other methods for typing of degraded DNA should be used, which are common in the field of forensic research (Timken et al 2005;Meissner et al 2007).…”