Fluorescent postlabelling of modified DNA bases

Postlabelling of modified DNA bases by methods other than the 32P method has attracted little attention to date. Three approaches using fluorescence have been made: (a) detection of a characteristic adduct-DNA functionality, e.g. reaction of cis-vicinal diols of PAH-DNA adducts with a boronate-containing fluorescent molecule; (b) reaction of modified nucleotides with a reactive fluorescent molecule, e.g. detection of 8-HOdG-5'-monophosphate by reaction with dansyl chloride; (c) reaction of modified DNA bases with reagents giving fluorescent products, e.g. derivatization of 7-MeGua with phenylmalondialdehyde (PhMal). Recent results in developing the latter approach are described. Reaction of a series of 7-alkylguanines, including 7-methyl-, 7-ethyl-, 7-(2-hydroxyethyl)-, 7-carboxymethyl-, 7-(2-carboxyethyl)- and 7-(2,3-dihydroxypropyl)-guanine, with PhMal gave fluorescent products showing similar spectral properties. The detection limit for fluorescence detection following reverse-phase HPLC separation was typically less than 1 pmol injected. In order to improve the sample clean-up before analysis, an antiserum has been prepared against the fluorescent derivative of 7-(2-carboxyethyl)guanine bound to carrier protein via the carboxyl group. The resulting antiserum cross-reacted with a number of PhMal derivatives.