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© 1994 Oxford University Press

research-article

Optimization of 32P-postlabelling assays for the quantitation of O6-mettyl and N7-methyldeoxyguanosine-3' -monophosphates in human DNA

Kemal Haque, Donald P. Cooper and Andrew C. Povey 1

Cancer Research Campaign Department of Carcinogenesis, Paterson Institute for Cancer Research Manchester M20 9BX, UK

1To whom correspondence should be addressed

The 3' and 5'-monophosphates of O6-methyldeoxyguanosine and N7-methyldeoxyguanoslne were chemically synthesized. Using these standards with deoxyguanoslne-3'-monophosphate (dGp) as an internal standard, conditions were optimized to quantify O6 -methyldeoxyguanosine-3'-monophosphate (O6 and N7-methyldeoxyguanosine-3'-monophosphate (N7-MedGp) by 32P-postlabelling Under optimal conditions, the labelling efficiencies of O6 -MedGp and N7-MedGp were respectively {bsim}100 and {bsim}15%, with detection limits of {bsim}1.1 and {bsim}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 isolated from DNA digests by immnunoaffinity separation. The standard curve generated from the use of [3H]-O6MedGp, thus isolated, was identical to that generated previously using the chemically synthesized [3H]-O6-MedGp, indicating that no inhibitory factors co-eluted with the [3H]-O6-MedGp After passage through two immuno columns, recovery of 4 and 40 fmol of [3H]-O6-MedGp was {bsim}30%. Four human stomach samples were analysed by combining this immunoaffinity purification with 32P-post-labelling: levels ranged from 0.21 to 0.86 µmol 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 32 Whereas O6-MedGp was detected at levels between 0.3 and 3.4 µmol O6-MedGp/mol dG, no N7-MedGp was detected in these samples, probably due to depunnation of N7-MedGp to N7-methylguanine or reduced assay sensitivity resulting from contaminating nucleotides and/or unidentified radioactivity elating close to the N7-methyldeoxyguanosine-5'-monophosphate.


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Home page
 MutagenesisHome page
D.H. Phillips and M. Castegnaro
Standardization and validation of DNA adduct postlabelling methods: report of interlaboratory trials and production of recommended protocols
Mutagenesis, May 1, 1999; 14(3): 301 - 315.
[Abstract] [Full Text] [PDF]


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