Real-time quantitative PCR has become a widely used technique for sensitive and specific quantification of both human nuDNA and mtDNA in forensics and ancient DNA studies, offering several advantages with respect to other current methodologies (hybridization or end-point PCR methods), including: higher sensitivity and dynamic range of quantitation, unnecessary post-PCR processing, automation feasibility and high throughput, and the possibility to simultaneously perform different qualitative analyses (gender determination, mtDNA degradation, Taq inhibition rate, etc.).
However, the data obtained by comparing different real-time PCR methods across different laboratories (Kline et al., 2005) show differences with regard to precision and bias. These results emphasize the need to develop a standard reference material for DNA quantification in the forensic field (Kline et al., 2005).
Real-time PCR methods will continue to take advantage of technical developments to improve the multiplexing capability, the automation feasibility and the detection limit.
The use of real-time quantitative PCR methods for body fluid (saliva, semen and blood) identification by targeting messenger RNA markers (Nussbaumer et al., 2006) is a novel molecular approach within the forensic field that will probably increase in use and importance. The forensic application of real-time quantitative PCR assays to target other markers of gene expression remains to be explored.
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