The specific quantification of human DNA from forensic evidentiary materials is a recommended procedure for a reliable short tandem repeat (STR) analysis, which is the current gold standard for DNA profiling in forensic casework (Jobling and Gill, 2004) (see Chapter 5). An estimation of the DNA quantity is made to adjust the DNA input of subsequent end-point polymerase chain reaction (PCR)-based DNA profiling methods, preventing PCR failures that are due to the absence of DNA, and avoiding PCR artefacts such as random allele dropout produced by stochastic amplification effects from low copy number (LCN) DNA samples (under 100 pg of DNA) (Gill et al., 2000), and also preventing off-scale over-amplification artefacts (including n + 1 peaks, increased stutter bands and pullout) associated with an excess of DNA input in the PCR. In addition, an accurate DNA quantification helps to prevent the unnecessary waste of DNA, which is especially important when analysing LCN DNA samples.
In the past, forensic laboratories have used the slot-blot hybridization approach to target the D17Z1 locus (Waye and Willard, 1986) - a highly repetitive alphoid primate-specific sequence - for DNA quantification in forensic casework (Waye et al., 1989). However, this methodology, with a detection limit above the limit of the STR profiling approaches, was often not sensitive enough to detect LCN forensic DNA samples. In addition, the method is labour-intensive, time-consuming and poorly suited to high-throughput sample flow.
Several studies (Andreasson et al., 2002; Alonso et al., 2003; Nicklas and Buel, 2003; Richard et al., 2003; Walker et al., 2003, 2005; Alonso et al., 2004;
Molecular Forensics. Edited by Ralph Rapley and David Whitehouse Copyright 2007 by John Wiley & Sons, Ltd.
Timken et al., 2005; Andreasson et al., 2006; Swango et al., 2006) have demonstrated the usefulness of real-time PCR for a sensitive, specific and high-throughput quantification of both human nuclear DNA (nuDNA) and mitochondrial DNA (mtDNA) in forensics and ancient DNA studies.
The recent development of a commercially available real-time PCR human DNA quantification kit (Green et al., 2005) has also contributed to a worldwide use of real-time PCR in forensic genetics.
In this chapter we will review all the different real-time PCR assays that have been applied in forensic genetics for the specific quantification of human autosomal, X and Y chromosome and mitochondrial DNA targets. We also review some real-time PCR assays for rapid quantification of non-human species of interest in forensic and ancient DNA studies, including the identification of pathogens in microbial forensics (Budowle et al., 2005). Other applications of real-time PCR assays, such as allele discrimination and single nucleotide polymorphism genotyping, are outside of the scope of this review.
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