Simultaneous determination of pyrimidine or purine deoxyribonucleoside triphosphates using a polymerase assay.
In this paper, we describe an improved enzymatic assay for the determination of deoxyribonucleoside triphosphates (dNTPs). This is based on the elongation of 32P 5'-end-labeled oligonucleotide primers annealed to complementary 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 limiting. 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 allows for the first time the simultaneous determination of two pyrimidine or two purine deoxyribonucleoside triphosphates. Consequently, a large number of biological samples can be tested in a single experiment. The high sensitivity of this method enables the quantification of low concentrations of dNTPs, such as those found in resting nondividing cells. Furthermore, this new protocol is well suited for the determination of dNTPs in cells treated with the antiretroviral ddI, since the Klenow fragment has a low affinity for ddATP, the active form of ddI.
Roy
B
B
Institut de Biochemie Biophysique Cellulaire et Moléculaire Unité CNRS 571, Université Paris-Sud, Orsay, France. beatrice.roy@bbmpc.u-psud.fr
Beuneu
C
C
Roux
P
P
Buc
H
H
Lemaire
G
G
Lepoivre
M
M
D019380:Anti-HIV Agents / Q000032:analysis / Q000378:metabolism; D001483:Base Sequence; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D004256:DNA Polymerase I; D017931:DNA Primers / Q000235:genetics; D003838:Deoxyadenine Nucleotides / Q000032:analysis / Q000378:metabolism; D003854:Deoxyribonucleotides / Q000032:analysis*; D016049:Didanosine / Q000378:metabolism; D054306:Dideoxynucleotides; D006801:Humans; D008214:Lymphocytes / Q000737:chemistry; D012680:Sensitivity and Specificity
D019380:Anti-HIV Agents; D017931:DNA Primers; D003838:Deoxyadenine Nucleotides; D003854:Deoxyribonucleotides; D054306:Dideoxynucleotides; C037773:2',3'-dideoxyadenosine triphosphate; D004256:DNA Polymerase I; D016049:Didanosine