Optimization of 32P-postlabelling assays for the quantitation of O6-methyl and N7-methyldeoxyguanosine-3'-monophosphates in human DNA.
AffiliationCancer Research Campaign Department of Carcinogenesis, Paterson Institute for Cancer Research, Manchester, UK.
MetadataShow full item record
AbstractThe 3' and 5'-monophosphates of O6-methyldeoxyguanosine and N7-methyldeoxyguanosine were chemically synthesized. Using these standards with deoxyguanosine-3'-monophosphate (dGp) as an internal standard, conditions were optimized to quantify O6-methyldeoxyguanosine-3'-monophosphate (O6-MedGp) and N7-methyldeoxyguanosine-3'-monophosphate (N7-MedGp) by 32P-postlabelling. Under optimal conditions, the labelling efficiencies of O6-MedGp and N7-MedGp were respectively approximately 100 and approximately 15%, with detection limits of approximately 1.1 and approximately 6.0 fmol respectively using 10 pmol dGp or 0.8 fmol of O6-MedGp if 2 pmol of dGp was used. The assay developed for O6-MedGp was then applied to the quantitation of [3H]-O6-MedGp and O6-MedGp isolated from DNA digests by immunoaffinity separation. The standard curve generated from the use of [3H]-O6-MedGp, thus isolated, was identical to that generated previously using the chemically synthesized O6-MedGp, indicating that no inhibitory factors co-eluted with the O6-MedGp. After passage through two immunocolumns, recovery of 4 and 40 fmol of O6-MedGp was approximately 30%. Four human stomach samples were analysed by combining this immunoaffinity purification with 32P-post-labelling: levels ranged from 0.21 to 0.86 mumol O6-MedGp/mol dG. Further DNA samples, isolated from the human colon, were fractionated by anion-exchange HPLC and the N7-MedGp and O6-MedGp containing fractions were purified by reverse-phase HPLC and immunoaffinity chromatography respectively. Adduct-containing fractions were dried and 32P-postlabelled. Whereas O6-MedGp was detected at levels between 0.3 and 3.4 mumol O6-MedGp/mol dG, no N7-MedGp was detected in these samples, probably due to depurination of N7-MedGp to N7-methylguanine or reduced assay sensitivity resulting from contaminating nucleotides and/or unidentified radioactivity eluting close to the N7-methyldeoxyguanosine-5'-monophosphate.
CitationOptimization of 32P-postlabelling assays for the quantitation of O6-methyl and N7-methyldeoxyguanosine-3'-monophosphates in human DNA. 1994, 15 (11):2485-90 Carcinogenesis
- Accurate and sensitive quantitation of N7-methyldeoxyguanosine-3'-monophosphate by 32P-postlabeling and storage-phosphor imaging.
- Authors: Haque K, Cooper DP, van Delft JH, Lee SM, Povey AC
- Issue date: 1997 Jun
- The development, validation and application of a 32P-postlabelling assay to quantify O6-methylguanine in human DNA.
- Authors: Povey AC, Cooper DP
- Issue date: 1995 Jul
- Detection of N7-methyldeoxyguanosine adducts in human pulmonary alveolar cells.
- Authors: Petruzzelli S, Tavanti LM, Celi A, Giuntini C
- Issue date: 1996 Aug
- Associations between smoking, GST genotypes and N7-methylguanine levels in DNA extracted from bronchial lavage cells.
- Authors: Lewis SJ, Cherry NM, Niven RM, Barber PV, Povey AC
- Issue date: 2004 Apr 11
- Formation and persistence of N7-methylguanine DNA adducts in the target pyloric tissue following chronic exposure to N-methyl-N'-nitro-N-nitrosoguanidine.
- Authors: Haque K, Cooper DP, Povey AC
- Issue date: 1999