There are two general fluorogenic methods to monitor the real-time progress of the PCR: by measuring Taq polymerase activity using double-stranded DNA binding dye chemistry (SYBR Green or ethidium bromide) (Higuchi et al., 1993) or by measuring the 5'-nuclease activity of the Taq DNA polymerase to cleave a target-specific fluorogenic probe (a TaqMan probe: an oligonucleotide complementary to a segment of the template DNA, with both a reporter and a quencher dye attached, that only emits its characteristic fluorescence after cleavage) (Holland et al., 1991) Although TaqMan assays are the most popular probe-based assays in forensic laboratories, alternative probe-based chemistry, such as molecular beacons (Tyagi and Kramer, 1996) or scorpion primers and probes (Whitcombe et al., 1999), could also be employed for specific target detection.
Real-time analysis of the fluorescence levels at each cycle of the PCR (amplification plot) allows a complete picture to be obtained of the whole amplification process for each sample. In the initial cycles of the PCR a baseline is observed without any significant change in fluorescence signal. An increase in fluorescence above the baseline indicates the detection of accumulated PCR product. The higher the initial input of the target genomic DNA, the sooner a significant increase in fluorescence is observed. The cycle at which fluorescence reaches an arbitrary threshold level during the exponential phase of the PCR is named Ct (threshold cycle). A standard curve can be generated by plotting the log of the starting DNA template amount of a set of previously quantified DNA standards against their Ct values. Therefore, an accurate estimation of the starting DNA amount from unknown samples is accomplished by comparison of the measured Ct values with the Ct values of the standard curve.
Compared to end-point PCR quantification methods, the use of Ct values is a more reliable quantification assay. This is mainly due to the fact that Ct determination is performed during the high-precision exponential phase of the PCR when none of the reaction components are limiting, contrary to PCR end-point measurements.
Although SYBR Green assay provides the simplest and most economical format for detecting and quantifying PCR products in real-time reactions, the main limitation is that non-specific amplifications (primer-dimer, non-human products, etc.) cannot be distinguished from specific amplifications. On the other hand, the amplicon/dye ratio varies with amplicon length. In addition, SYBR Green can only be used in single PCR reactions, therefore the use of this assay should be restricted to optimized PCR reactions producing single PCR products free from non-specific PCR artefacts.
The probe-based real-time PCR assay has been the method of choice to quantify human nuDNA and mtDNA in forensic genetics because of its superior specificity and quantitation accuracy in comparison with SYBR Green assays (see Chapter 9). Another advantage of probe-based methods is the feasibility to perform multiplex PCR of different targets (Andreasson et al., 2002; Timken et al., 2005; Walker et al., 2005). Probe-based assays also provide the possibility to perform, in a single PCR reaction, not only specific human DNA quantification but also different qualitative analyses, such as gender determination (Alonso et al., 2003, 2004; Andreasson and Allen, 2003; Green et al., 2005), DNA degradation (Alonso et al., 2004; Swango et al., 2006) and Taq inhibition rate (Timken et al., 2005; Green et al., 2006; Swango et al., 2006).
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