Escherichia coli DNA polymerase III epsilon subunit increases Moloney murine leukemia virus reverse transcriptase fidelity and accuracy of RT-PCR procedures

Abstract

In an effort to improve reverse transcriptase (RT) fidelity, we measured the error rate of Moloney murine leukemia virus (MMLV) RT in the presence of several autonomous and DNA polymerase-associated 3'-5' exonucleases using a lacZ forward mutation assay. A number of 3'-5' exonucleases were found to lower the error rate of MMLV RT, including p53, Escherichia coli DNA polymerase III epsilon subunit, and the proofreading activities associated with T4, varphi29, and E. coli pol I DNA polymerases. The bacterial epsilon subunit increased RNA-dependent DNA synthesis fidelity by approximately threefold and was the only 3'-5' exonuclease tested that did not deleteriously affect RT-PCR yields. Further testing showed that RT-PCR mutant frequencies were reduced significantly by performing cDNA synthesis in the presence of epsilon subunit, followed by PCR with a high-fidelity proofreading DNA polymerase. DNA sequence analysis was used to show that the combination of MMLV RT/epsilon subunit and PfuUltra DNA polymerase produces approximately eightfold fewer errors compared with the commonly used combination of MMLV RT and a Taq-based high-fidelity blend, consistent with predictions based on experimentally determined polymerase error rates.