Characterization of a novel DNA polymerase activity assay enabling sensitive, quantitative and universal detection of viable microbes.

ABSTRACT: During the past 50 years, in vitro measurement of DNA polymerase activity has become an essential molecular biology tool. Traditional methods used to measure DNA polymerase activity in vitro are undesirable due to the usage of radionucleotides. Fluorescence-based DNA polymerase assays have been developed; however, they also suffer from various limitations. Herein we present a rapid, highly sensitive and quantitative assay capable of measuring DNA polymerase extension activity from purified enzymes or directly from microbial lysates. When tested with purified DNA polymerase, the assay detected as little as 2 × 10(-11)U of enzyme (∼ 50 molecules), while demonstrating excellent linearity (R(2)=0.992). The assay was also able to detect endogenous DNA polymerase extension activity down to less

SUBMITTER: Zweitzig DR 

PROVIDER: S-EPMC3413125 | biostudies-literature | 2012 Aug

REPOSITORIES: biostudies-literature