The use of a TaqMan real-time PCR assay for quantification of mitochondrial human DNA from forensic specimens was first described by Andreasson et al. (2003) by targeting a 142 bp region spanning over the genes for tRNA lysine and ATP Synthase 8 that can be amplified in a single PCR reaction or in combination with an nuDNA target. The assay was tested on 236 forensic specimens (hair, bloodstains, fingerprints, skin debris, saliva stains and others) containing from zero to >100 000 mtDNA copies and more recently on different LCN DNA samples such as fingerprints (Andreasson et al., 2006).
Alonso et al. (2003, 2004) reported the specific quantification of human mtDNA by monitoring the real-time progress of the PCR amplification of two different fragment sizes (113 bp and 287 bp) within the hypervariable region 1 (HV1) of the mtDNA control region, using two fluorogenic probes to specifically determine the mtDNA copy of each fragment size category. This additional information - number of copies in each size category - has been demonstrated to be very helpful to evaluate the mtDNA preservation state from ancient bone samples.
A 69 bp fragment of the mtDNA NADH dehydrogenase subunit 1(ND1) locus has also been used as a target for mtDNA quantitation in forensic specimens using a TaqMan duplex real-time PCR assay that allows simultaneous quantification of human nuDNA (Timken et al., 2005). Another mtDNA target used in forensics by TaqMan-MGB assays is a 79 bp fragment of a conserved region of the human mtDNA, which is co-amplified with an autosomal and a Y chromosome target (Walker et al., 2005).
Recently a novel forensic use of quantitative real-time PCR (rtPCR) using TaqMan-MGB probes has been described, targeting the highly variable mito-chondrial single nucleotide polymorphism 16519T/C to investigate heteroplas-mic mixtures with an accurate quantification of the minor allele down to 9% (Niederstatter et al., 2006).
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