32P-Postlabeling Analysis of DNA Adducts.

32P-Postlabeling analysis, originally described by Kurt Randerath and colleagues, is an ultra-sensitive method for the detection and quantitation of DNA adducts, covalent modifications of the DNA. It consists of four main steps: Enzymatic digestion of DNA to 3'-monophosphate nucleosides (modified or unmodified) using micrococcal nuclease (endonuclease) and spleen phosphodiesterase (exonuclease); enrichment of the adducts by nuclease P1 digestions or butanol extraction prior to labeling; 5'OH-radiolabeling of the adducts by T4 polynucleotide kinase-catalyzed transfer of 32P-orthophosphate from [γ-32P]ATP; chromatographic separation of labeled adducts by thin-layer chromatography and detection and quantification by means of their radioactive decay. Adducted nucleotide-3'-5'-bisphosphates are then separated from their normal counterparts by multidirectional thin-layer chromatography. The assay requires only micrograms of DNA and is capable of detecting adduct levels as low as 1 adduct in 109-1010 normal nucleotides. It is applicable to a wide range of investigations in human, animal, and in vitro studies including monitoring exposure to environmental or occupational carcinogens, determining whether a chemical or a complex mixture has genotoxic properties, elucidation of the toxicological pathways of carcinogens, and monitoring DNA repair.