The effects of nested primer binding sites on the reproducibility of PCR: mathematical modeling and computer...

The polymerase chain reaction (PCR) has become an indispensable tool in modern biological research. Although the application of PCR is a standard routine, we widely lack a theoretical understanding of the dynamic processes involved, especially with respect to the amplification of nonreproducible and/or unexpected amplification products. For one potential source of uncertainty, the presence of nested primer binding sites within an amplifyable DNA locus, we consider a simple stochastic model for the dynamics of PCR amplification of competing products. For commonly used thermostable DNA polymerases lacking a 5'-3'-exonuclease activity, we predict the relative amplification frequencies of competing PCR products dependent on the primer binding probability, the number of PCR cycles, and the number of initial DNA template molecules. At low primer binding probabilities and low numbers of initial DNA template molecules and PCR cycles, we expect the amplification of two products. At high primer binding probabilities and/or high copy numbers of initial template molecules only one main amplification product is predicted at increasing cycle numbers. Furthermore, by means of computer simulation studies we quantify the stochastic variation for the amplification frequencies of competing products.