Add time:09/08/2019         Source:sciencedirect.com

A new method is presented for the determination of 2′-deoxythymidine 5′-triphosphate and 2′-deoxycytidine 5′-triphosphate concentrations within human cells based on a DNA polymerase reaction directed by a palindromic oligonucleotide precursor. Two 19-mer oligonucleotide precursors are employed that contain a common 8-mer palindromic sequence followed by a sequence-specific insertion site and a 5′-oligodeoxythymidylate tail. To conduct a measurement, two molecules of the 19-mer oligonucleotide precursor are first annealed to form a pair of symmetrical template–primer addition sites at their 3′-termini that are coded for the analyte of interest, present in limiting amounts. The Klenow fragment ofEscherichia coliDNA polymerase I then elongates the template–primer by the addition of two molecules of the complementary deoxyribonucleotide analyte. Following the addition of the analyte molecules, the template–primer is extended with a 10-mer oligo(dA) tail in the presence of excess dATP and the Klenow fragment. The result is a 30-mer palindromic 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 oligonucleotide 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 analytes in as few as 2.5 × 106human cells are presented.

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