M. Dusinska et A. Collins, DETECTION OF OXIDIZED PURINES AND UV-INDUCED PHOTOPRODUCTS IN DNA OF SINGLE CELLS, BY INCLUSION OF LESION-SPECIFIC ENZYMES IN THE COMET ASSAY, ATLA. Alternatives to laboratory animals, 24(3), 1996, pp. 405-411
The comet assay (single cell gel electrophoresis) is a rapid, very sen
sitive method for the detection of DNA strand breaks at the level of s
ingle cells, which is now being applied in genotoxicity testing. We mo
dified this method for the detection of a variety of kinds of DNA lesi
on, by treating nucleoid DNA in the gel with either formamidopyrimidin
e-DNA glycosylase (which recognises ring opened purines, 8-hydroxyguan
ine and apurinic/apyrimidinic sites), or uvrABC excinuclease (uvrABC;
which has a rather broad specificity, including bulky lesions and UV p
hotoproducts). By using this modified assay, we demonstrate the remova
l of DNA strand breaks and oxidised purines upon incubating cells afte
r treatment with hydrogen peroxide. This modification clearly increase
s the usefulness of the assay for the analysis of DNA damage and repai
r, for screening human populations for DNA damage, and for testing nov
el chemicals for genotoxicity.