Accuracy of replication in the polymerase chain reaction. Comparison between Thermotoga maritima DNA polymerase and Thermus aquaticus DNA polymerase.

R.S Diaz,E.C Sabino

doi:10.1590/s0100-879x1998001000001

Abstract

For certain applications of the polymerase chain reaction (PCR), it may be necessary to consider the accuracy of replication. The breakthrough that made PCR user friendly was the commercialization of Thermus aquaticus (Taq) DNA polymerase, an enzyme that would survive the high temperatures needed for DNA denaturation. The development of enzymes with an inherent 3' to 5' exonuclease proofreading activity, lacking in Taq polymerase, would be an improvement when higher fidelity is needed. We used the forward mutation assay to compare the fidelity of Taq polymerase and Thermotoga maritima (ULTMA) DNA polymerase, an enzyme that does have proofreading activity. We did not find significant differences in the fidelity of either enzyme, even when using optimal buffer conditions, thermal cycling parameters, and number of cycles (0.2% and 0.13% error rates for ULTMA and Taq, respectively, after reading about 3,000 bases each). We conclude that for sequencing purposes there is no difference in using a DNA polymerase that contains an inherent 3' to 5' exonuclease activity for DNA amplification. Perhaps the specificity and fidelity of PCR are complex issues influenced by the nature of the target sequence, as well as by each PCR component.

Highlights

In the present study we compared the accuracy of replication in polymerase chain reaction (PCR) between Thermus aquaticus (Taq) polymerase and Thermotoga maritima (ULTMATM) DNA polymerase by DNA sequencing
Biochemical fidelity measurements of single nucleotide incorporation/misincorporation have indicated that the ability of “nonproofreading” DNA polymerases (AMV reverse transcriptase and D. megalanogaster DNA polymerase) to misincorporate a deoxynucleotide triphosphate is critically determined by the concentration of the triphosphate [6]
Other conditions known to reduce the fidelity of Taq polymerase in vivo are high MgCl2 in the presence of MnCl2, and high number of PCR cycles starting with low target input [8]

Summary

Introduction

In the present study we compared the accuracy of replication in PCR between Thermus aquaticus (Taq) polymerase and Thermotoga maritima (ULTMATM) DNA polymerase by DNA sequencing. Differences in fidelity during amplification can lead to differences in the fraction of PCR products with a sequence identical to the original target. Each polymerase-induced error, once introduced, will be amplified exponentially along with the original wild type sequences during subsequent cycles.

Results

Conclusion