Optimization of the polymerase chain reaction with regard to fidelity: Modified T7, Taq, and vent DNA polymerases

Abstract

The fidelity of DNA polymerases used in the polymerase chain reaction (PCR) can be influenced by many factors in the reaction mixture. To maximize the fidelity of DNA polymerases in the PCR, pH, concentrations of deoxynucleoside triphosphates, and magnesium ion were varied. Denaturing gradient gel electrophoresis was used to separate the polymerase-induced mutants from wild-type DNA sequences. Thermolabile modified T7 DNA polymerase, thermostable Taq, and Vent DNA polymerases were studied. Fidelity of all three DNA polymerases was sensitive to concentrations of deoxynucleoside triphosphates, magnesium ion, and pH. Within conditions that permitted efficient amplification, optimization with regard to these three factors yielded an average error rate in error/base pair incorporated of 7.2 × 10-5 for Taq, 4.5 × 10-5 for Vent, and 4.4 × 10-5 for modified T7 (Sequenase) DNA polymerases.