A Dot-Blot Immunoassay for Measuring Repair of Ultraviolet Photoproducts

The method described here makes use of a polyclonal antiserum to measure repair of the principal photoproducts induced in DNA by short-wave ultraviolet light (UV-C)—pyrimidine-pyrimidone 6-4 photoproducts ([6-4]PPs) and cyclobutane pyrimidine dimers (CPDs). DNA extracted from irradiated cells is applied to a nitrocellulose dot-blot and quantified using an enzyme-conjugated secondary antibody and a color assay. Though the polyclonal antiserum contains antibodies to both cyclobutane pyrimidine dimers and (6-4) photoproducts, repair of these can be measured separately by differential destruction of one or other photoproduct. The method is useful for measuring repair in total genomic DNA. It is more sensitive than most other methods and is sufficiently sensitive to measure repair of damage induced by doses of 10 J/m² of UV-C in DNA from mammalian cells.