PALINDROMIC OLIGONUCLEOTIDE-DIRECTED ENZYMATIC DETERMINATION OF 2'-DEOXYTHYMIDINE 5'-TRIPHOSPHATE AND 2'-DEOXYCYTIDINE 5'-TRIPHOSPHATE IN HUMAN-CELLS

A new method is presented for the determination of 2'-deoxythymidine 5 '-triphosphate and 2'-deoxycytidine 5'-triphosphate concentrations wit hin human cells based on a DNA polymerase reaction directed by a palin dromic oligonucleotide precursor. Two 19-mer oligonucleotide precursor s are employed that contain a common 8-mer palindromic sequence follow ed by a sequence-specific insertion site and a 5'-oligodeoxythymidylat e tail, To conduct a measurement, two molecules of the 19-mer oligonuc leotide precursor are first annealed to form a pair of symmetrical tem plate-primer addition sites at their 3'-termini that are coded for the analyte of interest, present in limiting amounts. The Klenow fragment of Escherichia coli DNA polymerase I then elongates the template-prim er by the addition of two molecules of the complementary deoxyribonucl eotide analyte. Following the addition of the analyte molecules, the t emplate-primer is extended with a 10-mer oligo(dA) tail in the presenc e of excess dATP and the Klenow fragment. The result is a 30-mer palin dromic oligonucleotide that can be separated from any remaining 19-mer precursor and quantified by paired-ion HPLC using UV detection, Since the molar extinction coefficient of the 30-mer palindromic oligonucle otide is much larger than that of the nucleotide analyte alone, the UV signal is markedly enhanced, thereby increasing sensitivity. Details describing this method and the application of it to measure these anal ytes in as few as 2.5 x 10(6) human cells are presented.