Molecular Toxicology Protocols pp 551-564
An Immunoassay for Measuring Repair of UV Photoproducts
A method is described that makes use of a polyclonal antiserum to measure repair of the principal photoproducts induced in DNA by short-wave ultraviolet light (UVC)—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 quantitated using an enzyme-conjugated secondary antibody and a color assay. Although the polyclonal antiserum contains antibodies to both [6-4]PPs and CPDs, repair of these lesions can be measured separately by differential destruction or repair of one or other photoproduct. The method is useful for measuring repair in total genomic DNA, and is sufficiently sensitive to measure repair of damage induced by doses of 10 J/m2 of UVC and less. The method is very versatile and has been used to measure repair in human cells, yeasts, plants, archaea, bacteria, and filamentous fungi.
Key wordsUV damage DNA repair Cyclobutane dimmers (6-4) Photoproducts Immunoassay Dot blot
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