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. Use of PCR primers tagged with a fluorescent dye that acts as a reporter allows the quantitative progress of a PCR amplification experiment to be monitored in “real time.” The number of 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 inflection points (slopes) of the curves of two reactions with known initial concentrations (10 or 100,000 copies: square and diamond] can be used to draw a standard curve [right], which allows the number of gene copies in the 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.

All figure & material ©2013 by  Steven M. Carr