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 x 10(-5) for Taq,
4.5 x 10(-5) for Vent, and 4.4 x 10(-5) for modified T7 (Sequenase)
DNA polymerases.
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