Open AccessAn Enzymatic Method for the Determination of dCTP and dGTP in Picomole Amounts
Author(s) -
Skoog Lambert
Publication year - 1970
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1970.tb01154.x
Subject(s) - nucleoside triphosphate , chemistry , limiting , enzyme , nucleoside , polymerase , biochemistry , nucleotide , adenosine triphosphate , dna , chromatography , gene , mechanical engineering , engineering
A rapid and accurate method for the measurement of picomole amounts of dCTP and dGTP is described. The method is based upon the ability of DNA‐polymerase, in the presence of poly[d(I‐C)] primer template, to incorporate dCTP and dGTP into an acid‐insoluble product. When either dCTP or dGTP is the limiting factor in the reaction, the extent of polymerization can be measured by the extent of incorporation of complementary radioactively labeled excess nucleoside triphosphate into an acid‐insoluble product. The incorporation of radioactivity was found to be directly proportional to the amount of the limiting nucleoside triphosphate. It was possible to measure as little as 0.2 pmole of either nucleoside triphosphate. The optimal conditions for the assay were determined in terms of pH, magnesium requirement, poly[d(I‐C)] concentration, and enzyme concentration.