Immunochemical detection of DNA in alkaline sucrose gradient fractions.
A procedure for the detection of non-radioactive DNA in alkaline sucrose gradients is described. This method consists of the following steps: (1) fractionation of the DNA-containing gradients into microtiter plates, neutralization and overnight adsorption; (2) covalent labelling of the guanine bases of the adsorbed DNA by reaction with N-acetoxy-2-acetylaminofluorene; and (3) determination of the gradient profile by means of an enzyme-linked immunosorbent assay using antibodies with high affinity for dG-AAF. The method has been found suitable for the rapid and sensitive determination of gamma-ray-induced single-strand breaks and UV-induced pyrimidine dimers in mammalian cells in vitro. In addition, it has been shown that the method can be used for the determination of pyrimidine dimers induced in skin cells in vivo.
D000099:Acetoxyacetylaminofluorene; D000818:Animals; D002478:Cells, Cultured / Q000528:radiation effects; D002499:Centrifugation, Density Gradient / Q000379:methods*; D004247:DNA / Q000032:analysis* / Q000276:immunology / Q000528:radiation effects; D004307:Dose-Response Relationship, Radiation; D005260:Female; D005720:Gamma Rays; D011740:Pyrimidine Dimers / Q000032:analysis; D051381:Rats; D014466:Ultraviolet Rays
D011740:Pyrimidine Dimers; D000099:Acetoxyacetylaminofluorene; D004247:DNA