A kinetic-based sigmoidal model for the polymerase chain reaction and its application to high-capacity absolute quantitative real-time PCR-1

En I. Each amplification reaction contained 100 femtograms of lambda gDNA (1,876 genomes) and 500 μM of the primers K7B and K12. The gain setting for each run was adjusted to ensure that reaction fluorescence remained below the saturation level of the photomultiplier tube (about 40,000 FU). , and Screen shots of amplification profiles generated by the Stratagene MxPro qPCR software. This reveals that fluorescence intensity is dependent on SYBR Green I quantity, reflected by the large increase in the height of the amplification profiles as SYBR Green I quantity is increased. , and LRE plot of each respective amplification profile. This reveals a linear domain corresponding to the central region of each amplification profile, confirming the mathematical prediction that amplification efficiency is linearly coupled to amplicon quantity. A consecutive group of points was selected (designated by red circles) for linear regression analysis (referred to as "LRE analysis"), generating estimates for (intercept) and (slope), from which was calculated using equation 4 (see Figure 1 for additional details). Details as to how the boundaries of the linear region were determined are described later in the study. : quantity of supplementary SYBR Green I, : linear regression correlation coefficient.<p><b>Copyright information:</b></p><p>Taken from "A kinetic-based sigmoidal model for the polymerase chain reaction and its application to high-capacity absolute quantitative real-time PCR"</p><p>http://www.biomedcentral.com/1472-6750/8/47</p><p>BMC Biotechnology 2008;8():47-47.</p><p>Published online 8 May 2008</p><p>PMCID:PMC2397388.</p><p>