THE EFFECTS OF NESTED PRIMER BINDING-SITES ON THE REPRODUCIBILITY OF PCR - MATHEMATICAL-MODELING AND COMPUTER-SIMULATION STUDIES

The polymerase chain reaction (PCR) has become an indispensable tool i n modern biological research, Although the application of PCR is a sta ndard routine, we widely lack a theoretical understanding of the dynam ic processes involved, especially with respect to the amplification of nonreproducible and/or unexpected amplification products, For one pot ential source of uncertainty, the presence of nested primer binding si tes within an amplifyable DNA locus, we consider a simple stochastic m odel 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 compet ing PCR products dependent on the primer binding probability, the numb er of PCR cycles, and the number of initial DNA template molecules, At low primer binding probabilities and low numbers of initial DNA templ ate molecules and PCR cycles, we expect the amplification of two produ cts, At high primer binding probabilities and/or high copy numbers of initial template molecules only one main amplification product is pred icted at increasing cycle numbers, Furthermore, by means of computer s imulation studies we quantify the stochastic variation for the amplifi cation frequencies of competing products.