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consists of two rounds of PCRs, where the quantify the initial amounts of template DNA
primers of the second PCR bind to an internal molecules with high accuracy over a wide
region of the first amplified DNA product. range of concentrations (Schmittgen et al.
Thus, the larger fragment produced by the 2000). Other advantages are the closed-tube
first round of PCR is used as the template for format, which reduces the risks of carryover
the second round. The specificity is particu- contamination, the fast and simple perfor-
larly enhanced because this technique helps mance, the wide dynamic range of quantifi-
to eliminate any spurious nonspecific ampli- cation (higher than six orders of magnitude),
fication product. However, the high risk of and the significantly higher reliability of the
cross contamination is the main drawback, results compared with conventional PCR.
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