P-32 POSTLABELING APPROACHES FOR THE DETECTION OF 8-OXO-2'-DEOXYGUANOSINE-3'-MONOPHOSPHATE IN DNA

P-32-Postlabelling methods have been investigated for the analysis of the oxidative DNA damage lesion 8-oxoguanine. The extent of digestion of commercially available calf thymus DNA and an 8-oxo-2'-deoxyguanosi ne-3'-monophosphate (8oxodGp) containing oligonucleotide to 2'-deoxynu cleotide-3'-monophosphates, using calf spleen phosphodiesterase and mi crococcal nuclease, was determined by HPLC. The extent of unmodified n ucleotide release from DNA, and the extent of 8oxodGp released from th e oligomer did not increase between 1 and 16 h of incubation at 37 deg rees C. Normal nucleotide release from DNA was found to be quantitativ e under these conditions, and 8oxodGp release from the oligomer was in the range of 84-91%. RNA contamination in DNA prepared for P-32-postl abelling severely compromised 8oxodGp analysis, Guanosine-3'-monophosp hate (Gp) was found to exhibit similar chromatographic and electrophor etic properties to 8oxodGp and as such compromised both 8oxodGp isolat ion in enrichment steps and subsequent resolution of the P-32-labelled bisnucleotides by TLC, The effect of ribonuclease A, T-1 and T-2 was investigated and a combination of A + T-1 was found to reduce Gp conta mination in DNA samples to levels which no longer interfered with 8oxo dGp analysis. We have successfully applied an HPLC enrichment protocol to the analysis of 8oxodGp in calf thymus DNA, Since determination of damage levels in human samples is often restricted by the amount of D NA available for analysis, a novel capillary electrophoresis (CE) tech nique for the enrichment of 8oxodGp has been developed, The advantage of CE is that it can achieve resolution of 8oxodGp and unmodified deox ynucleotides from much smaller samples and minimises the amount of [ga mma-P-32]ATP necessary for the analysis. (C) 1997 Elsevier Science B.V .