Mathematical model of real-time PCR kinetics
Article first published online: 16 SEP 2005
Copyright © 2005 Wiley Periodicals, Inc.
Biotechnology and Bioengineering
Volume 92, Issue 3, pages 346–355, 5 November 2005
How to Cite
Gevertz, J. L., Dunn, S. M. and Roth, C. M. (2005), Mathematical model of real-time PCR kinetics. Biotechnol. Bioeng., 92: 346–355. doi: 10.1002/bit.20617
- Issue published online: 13 OCT 2005
- Article first published online: 16 SEP 2005
- Manuscript Accepted: 16 MAY 2005
- Manuscript Received: 17 FEB 2005
- NSF CAREER Award. Grant Number: BES-0238617
- gene expression;
- mathematical modeling;
- PCR efficiency
Several real-time PCR (rtPCR) quantification techniques are currently used to determine the expression levels of individual genes from rtPCR data in the form of fluorescence intensities. In most of these quantification techniques, it is assumed that the efficiency of rtPCR is constant. Our analysis of rtPCR data shows, however, that even during the exponential phase of rtPCR, the efficiency of the reaction is not constant, but is instead a function of cycle number. In order to understand better the mechanisms belying this behavior, we have developed a mathematical model of the annealing and extension phases of the PCR process. Using the model, we can simulate the PCR process over a series of reaction cycles. The model thus allows us to predict the efficiency of rtPCR at any cycle number, given a set of initial conditions and parameter values, which can mostly be estimated from biophysical data. The model predicts a precipitous decrease in cycle efficiency when the product concentration reaches a sufficient level for template–template re-annealing to compete with primer-template annealing; this behavior is consistent with available experimental data. The quantitative understanding of rtPCR provided by this model can allow us to develop more accurate methods to quantify gene expression levels from rtPCR data. © 2005 Wiley Periodicals, Inc.