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Assay by inhibition of repair to measure O 6 ‐methylguanine in DNA
Author(s) -
Mironov Nikolai M.,
MartelPlanche Ghyslaine,
Wild Christopher P.
Publication year - 1994
Publication title -
environmental and molecular mutagenesis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1
H-Index - 87
eISSN - 1098-2280
pISSN - 0893-6692
DOI - 10.1002/em.2850230206
Subject(s) - dna , substrate (aquarium) , microbiology and biotechnology , detection limit , chemistry , adduct , dna repair , biology , chromatography , biochemistry , organic chemistry , ecology
A procedure for measuring the level of O 6 ‐me‐thylguanine (O 6 ‐meG) in DNA is described. Repair of 32 P‐oligodeoxynucleotides containing O 6 ‐meG adducts by O 6 ‐alkylguanine alkyltransferase (AGT) was performed in the presence of different quantities of DNA containing unknown concentrations of O 6 ‐meG. Each methylated DNA sample inhibited the repair of oli‐godeoxynucleotide substrate to an extent dependent upon O 6 ‐meG concentration. Each DNA sample tested at different concentrations in the assay therefore had a characteristic inhibition curve and could be compared to the curves generated using reference DNA samples of known O 6 ‐meG concentration. We report the method of calculation of the O 6 ‐meG level in a given DNA sample by comparison of its inhibition curve with that of reference DNAs. This method of calculation does not require a knowledge of the exact quantity of the labelled substrate or AGT used. The method requires only 0.1‐10 μg of DNA, with a limit of detection of 0.8 fmol of O 6 ‐meG per μg of DNA. © 1994 Wiley‐Liss, Inc.