Use of the recursion formula of the Gompertz function for the quantitation of PCR-amplified templates.
One common drawback of the currently used procedures to quantitate the polymerase chain reaction (PCR) is that the statistical evaluation of the experimental data depends on many, not just trivial, model assumptions. In the present study we report on an improvement in this crucial step of the quantitative PCR. The experimental design underlying the introduced method is exactly the same as in the case of the so-called PCR. However, by applying growth curve analysis based on the recursion formula of the Gompertz function the kinetics of the accumulation of the amplicon are estimated conjointly from data spanning both the and phases of the reaction. We demonstrate the method by determining the relative number of templates (a 206 bp segment spanning the exon 3 of the X-chromosomal murine Hprt-gene) contained in known orders of dilutions of DNA isolated from the spleen of the C57BL/6J-mouse. [32P]-dATP incorporation was used in duplicate experiments to quantify the amplicons as a function of amplification cycles. Our results: i) indicate that the accumulation of the PCR product as a function of PCR cycles follows a sigmoidal pattern compatible with the Gompertz growth model (P<0.0000001); ii) directly support the thesis that the kinetical pattern of accumulation of amplicons of a given DNA fragment does not depend on the number of corresponding DNA templates provided to the reaction; iii) permit a simple direct evaluation of the parallelity in the course of the accumulation of amplicons from different template numbers as a function of amplification cycles, which is a silent preposition in the evaluation of the so-called PCR; iv) allow an easy quantitation of the relative number of provided templates.
Schlereth
W
W
Department of Nuclear Medicine, University of Wurzburg, D-97080 Wurzburg, Germany.
Bassukas
I D
ID
Deubel
W
W
Lorenz
R
R
Hempel
K
K
D005784:Gene Amplification; D008432:Mathematical Computing; D016133:Polymerase Chain Reaction / Q000379:methods*; D013698:Templates, Genetic