32P-Postlabelling approaches for the detection of 8-oxo-2′-deoxyguanosine-3′-monophosphate in DNA

Abstract

32P-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′-deoxyguanosine-3′-monophosphate (8oxodGp) containing oligonucleotide to 2′-deoxynucleotide-3′-monophosphates, using calf spleen phosphodiesterase and micrococcal nuclease, was determined by HPLC. The extent of unmodified nucleotide release from DNA, and the extent of 8oxodGp released from the oligomer did not increase between 1 and 16 h of incubation at 37°C. Normal nucleotide release from DNA was found to be quantitative under these conditions, and 8oxodGp release from the oligomer was in the range of 84–91%. RNA contamination in DNA prepared for 32P-postlabelling severely compromised 8oxodGp analysis. Guanosine-3′-monophosphate (Gp) was found to exhibit similar chromatographic and electrophoretic properties to 8oxodGp and as such compromised both 8oxodGp isolation in enrichment steps and subsequent resolution of the 32P-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 contamination in DNA samples to levels which no longer interfered with 8oxodGp 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 DNA available for analysis, a novel capillary electrophoresis (CE) technique for the enrichment of 8oxodGp has been developed. The advantage of CE is that it can achieve resolution of 8oxodGp and unmodified deoxynucleotides from much smaller samples and minimises the amount of [γ- 32P]ATP necessary for the analysis.