Determination of deoxyribonucleoside triphosphates using DNA polymerase: a critical evaluation

Abstract

There are more than twenty appreciably different deoxyribonucleoside triphosphate assays using DNA polymerase in the literature. Therefore, each aspect of this method has been critically evaluated, including the purity of the substrates and of DNA polymerase, the reaction conditions, product isolation, and the effect of cell extracts on the assay. The final method uses a phosphatase-free DNA polymerase preparation, 2'-dAMP to inhibit the 3' leads to 5' exonuclease of DNA polymerase I, and includes a correction of both the background incorporation and the sample incorporation for dilution of the specific activity of the radioactive precursor by the sample. The sensitivity, range, accuracy, and reproducibility are reported as well as values for the deoxyribonucleoside triphosphate content of Chinese hamster ovary K-1 cells.