Quantitative "Real-Time" (RT) PCR

          The Polymerase Chain Reaction (PCR) is ordinarily a preparative or qualitative procedure used to produce a large quantity of DNA for analysis. Quantitative PCR (qPCR) is an analytical procedure to determine how much DNA is present.

        A qPCR reaction besides the usual PCR components includes a single-stranded ssDNA "Reporter / Quencher" probe that binds to the 3' end of dsDNA template of interest.  When bound to the DNA strand, the "Quencher" prevents the "Reporter" from fluorescing. As the Taq polymerase extends the PCR copy, the Reporter end is displaced first, while the Quencher end remains bound. The Reporter is "un-quenched" and releases a quantum flash of light at a particular wavelength. The number of flashes recorded during each cycle equals the count of replications. Progress of the PCR is monitored in "real time" by the cumulative count of flashes.

    The number of PCR cycles required for the amplification to shift from the initial linear to the exponential phase is an indication of abundance of the original template [left]. The slope of a plot of the inflection points of the reaction curves for known initial concentrations (in the example, squares and diamonds for 10 and 100,000 initial copies] can be used to draw a log-linear standard curve [right]. This allows the number of initial gene copies in an unknown sample [triangle] to be calculated.

          Biodiversity applications: Use of species-specific PCR primer sequences would allow monitoring of any one species against a diverse background, for example, the relative abundance of Atlantic Cod in the stomach contents of Harp Seals.