Plant & Animal Genomes XIV Conference Plant & Animal Genomes XIV ConferenceJanuary 14-18, 2006
Town & Country Convention Center
San Diego, CA
Robert G. Rutledge , Donald Stewart
Utilizing linear regression analysis of fluorescence data produced by individual amplification reactions, a novel quantitative methodology is described that can generate variances that average less than +/-15%, over five magnitudes of target quantity. Founded upon a linear relationship between amplicon accumulation and loss in amplification rate, a linear equation was found not to only effectively predict entry into the plateau phase, but also allows determination of the amplification efficiency generated by individual amplification reactions. In addition to providing a linear representation of individual amplification reactions, the derivation of two novel mathematical functions further demonstrates that highly precise modeling of PCR amplification can be achieved, that in turn provides the foundation for the exceptional quantitative capabilities conferred by this kinetic-based methodology. Of practical importance, this approach does not require the construction of standard curves, thus abrogating the need to prepare quantified standards for each individual target. In addition to dramatically increasing the accuracy and reliability of real-time PCR quantification, elimination of standard curving has significant implications for the development of large-scale applications, such as is required for gene expression profiling. Combined with its amiability to automation, the computational simplicity of this kinetic-based approach provides an effective, easy to implement alternative to the threshold-based methods that currently predominate quantitative real-time PCR.