Simultaneous determination of pyrimidine or purine deoxyribonucleoside triphosphates using a polymerase assay

In this paper, we describe an improved enzymatic assay for the determinatio n of deoxyribonucleoside triphosphates (dNTPs). This is based on the elonga tion of P-32 5'-end-labeled oligonucleotide primers annealed to complementa ry oligonucleotide templates. Incorporation within the primer/template (p/t ) was catalyzed by the Klenow fragment of Escherichia coli DNA polymerase I under conditions where the concentration of the dNTP to be analyzed is lim iting. Using a combination of two different sized p/t pairs, dCTP and dTTP (or dATP and dGTP) were assayed together. Since the elongated products were clearly separated after electrophoresis on a denaturing 10% polyacrylamide gel, the two dNTPs could be quantified in a single lane. This method allow s for the first time the simultaneous determination of two pyrimidine or tw o purine deoxyribonucleoside triphosphates. Consequently, a large number of biological samples can be tested in a single experiment. The high sensitiv ity of this method enables the quantification of low concentrations of cNTP s, such as those found in resting nondividing cells. Furthermore, this new protocol is well suited for the determination of dNTPs in cells treated wit h the antiretroviral ddI, since the Klenow fragment has a low affinity for ddATP, the active form of ddI. (C) 1999 Academic Press.