IMPROVED HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHIC ANALYSIS OF INTRACELLULAR DEOXYRIBONUCLEOSIDE TRIPHOSPHATE LEVELS

The ability to measure intracellular deoxyribonucleoside triphosphate (dNTP) pool sizes is important for understanding the intracellular met abolism of DNA synthesis and repair. We have developed an improved met hod for measuring intracellular dNTP pool size by high-performance liq uid chromatography (HPLC). Previous methods have enabled accurate meas urement of dNTPs only in concentrations greater than approximately 10 pmol per 10(6) cells due to the inability to partially purify cell ext racts, to the inability to apply extracts from extremely large numbers of cells, to the lack of efficient columns, to the presence of incomp atible solvents, and to the inability to inject large volumes. We have modified a low-pressure strong anion-exchange column pre-step develop ed by others to concentrate and partially purify oxidized cell extract s while at the same time eluting them in a more compatible solvent for HPLC injection. The HPLC column is a YMC ODS-AQ column operating in a combined hydrophobic-interaction chromatography-reversed-phase chroma tography mode. The injection and elution solvents are both phosphate-b ased. Using this method it is possible to measure intracellular dNTP l evels well below 0.5 pmol per 10(6) cells or at the sensitivity of the DNA polymerase assay.