Simulation and real-time monitoring of polymerase chain reaction for its higher efficiency

Polymerase chain reaction (PCR) is an important molecular biological tool for the amplification of nucleic acids. PCR process can be divided into three phases according to the amplification rate: exponential, quasi-linear, and plateau. We investigated the cause of the plateau phenomenon through real-time monitoring of the amplification profile and computerized simulation. Possible limiting components, such as Taq DNA polymerase, primer pair, and dNTPs were added during quasi-linear phase, after which the differences in the amplification profiles were monitored. Modeling and computerized simulations were performed to look into the complex mechanism of the reactions, such as renaturation of templates during temperature transition from denaturation to annealing step and effective enzyme concentration profiles within the cycle progress. The decrease of effective polymerase concentration due to heat inactivation and product accumulation caused the PCR plateau. Addition of polymerase during the quasi-linear phase could increase the final product amount; however, the PCR process still reached the plateau phase in spite of polymerase addition. Simulation results suggest that renaturation of templates before competitive annealing reaction and decrease of effective enzyme concentration by non-specific binding of polymerase to double-stranded DNA is the main contribution to plateau forming.