The development, validation and application of a 32P-postlabelling assay to quantify O6-methylguanine in human DNA.
In this study, a combined immunoaffinity purification/32P-postlabelling procedure has been used to quantify O6-methyldeoxyguanosine-3'-monophosphate (O6-MedGp) in human DNA. DNA digests are subjected to a two-stage immunopurification in which the acetone-eluted fraction from the first stage is reapplied to a second immunocolumn, and the O6-MedGp specifically eluted using O6-methylguanosine (O6-MerG). O6-MedGp is then 32P-postlabelled in the presence of deoxyinosine-3'-monophosphate (dIp) as internal standard, separated by two-dimensional TLC and levels of the adduct quantified using storage phosphor technology. The recovery of O6-MedGp at levels between 0.4 and 500 fmol was 61%. Analysis of human DNA samples indicated that < 1 fmol O6-methyldeoxy-guanosine-5'-monophosphate (O6-MepdG) could be detected with a high degree of precision (coefficient of variation < 12%) during a 2 h exposure to a storage phosphor screen. The assay was then applied to 25 human samples from three separate populations, one of which was exposed to methylating agent chemotherapy, for which O6-methyl-deoxyguanosine (O6-MedG) levels had already been quantified by HPLC/radioimmunoassay. The results indicated a high degree of correlation between the two assays (r = 0.99). O6-MedGp was detected in all the samples analysed with levels ranging from 0.026 to 23.2 mumol O6-MedGp/mol dG. The minimum amount of O6-MepdG detected was 0.2 fmol. As there was no detectable signal in the area to which O6-MepdG maps in negative control samples, a detection limit based upon the signal/noise ratio was impossible to quantify. However the limit of detection of the storage phosphor technology itself was estimated by quantifying a visually identifiable compound, which mapped to the same region. The amount of this compound was determined to be 32 +/- 27 amol (n = 5). If a similar amount of O6-MepdG was detected from 50 micrograms of DNA, and assuming that the labelling efficiency and recovery was similar to that found in this study, then this would correspond to an adduct level of approximately 3 nmol O6-MedGp/mol dG.
Povey
A C
AC
Department of Carcinogenesis, Paterson Institute for Cancer Research, Manchester, UK.
Cooper
D P
DP
D002855:Chromatography, Thin Layer; D004247:DNA / Q000032:analysis*; D004249:DNA Damage; D003849:Deoxyguanosine / Q000031:analogs & derivatives / Q000032:analysis; D005260:Female; D006147:Guanine / Q000031:analogs & derivatives* / Q000032:analysis; D006801:Humans; D007553:Isotope Labeling / Q000379:methods; D008297:Male; D009711:Nucleotides / Q000032:analysis; D010761:Phosphorus Radioisotopes; D015203:Reproducibility of Results
D009711:Nucleotides; D010761:Phosphorus Radioisotopes; D006147:Guanine; D004247:DNA; C015558:O(6)-methyl-2'-deoxyguanosine; C008449:O-(6)-methylguanine; D003849:Deoxyguanosine