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 assump tions. In the present study we report on an improvement in this crucia l step of the quantitative PCR. The experimental design underlying the introduced method is exactly the same as in the case of the so-called 'exponential' PCR. However, by applying growth curve analysis based o n the recursion formula of the Gompertz function the kinetics of the a ccumulation of the amplicon are estimated conjointly from data spannin g both the 'exponential' and 'plateau' phases of the reaction. We demo nstrate the method by determining the relative number of templates (a 206 bp segment spanning the exon 3 of the X-chromosomal murine Hprt-ge ne) contained in known orders of dilutions of DNA isolated from the sp leen of the C57BL/6J-mouse. [P-32]- dATP incorporation was used in dup licate experiments to quantify the amplicons as a function of amplific ation cycles. Our results: i) indicate that the accumulation of the PC R product as a function of PCR cycles follows a sigmoidal pattern comp atible with the Gompertz growth model (P<0.0000001); ii) directly supp ort the thesis that the kinetical pattern of accumulation of amplicons of a given DNA fragment does not depend on the number of correspondin g DNA templates provided to the reaction; iii) permit a simple direct evaluation of the parallelity in the course of the accumulation of amp licons from different template numbers as a function of amplification cycles, which is a silent preposition in the evaluation of the so-call ed 'exponential' PCR; iv) allow an easy quantitation of the relative n umber of provided templates.