DNA Polymerase Binding to the Primer/template Duplex Affects the Efficiency of PCR

Abstract

Polymerase chain reaction(PCR) for DNA and RNA amplifications in vitro has a profound impact on modern molecular biology. Since design of proper primers is crucial for the success in PCR,many parameters have been used for primer design. However,the effect of DNA polymerase binding to primer /template duplex on PCR efficiency was not taken into account in conventional primer design programs. To reveal whether or not the DNA polymerase-primer /template binding affects the PCR efficiency,here we built structural models for the Taq DNA polymerase in complex with different primer /template sequences,and designed PCR primers according to relative binding free energies calculated by MM /GBSA method. We verified our primer design approach using Human Serum Albumin(HSA) gene and Mycobacterium tuberculosis pyrF gene,and found that the PCR efficiencies of different designed primers for both tested genes correlated well with the calculated binding free energies. Our finding indicates clearly that the DNA polymerase-primer /template binding affects the PCR efficiency significantly. Thus,the calculated binding free energy could be used as a new parameter to design efficient PCR primers.