32P-postlabeling analysis of DNA adducts

Abstract

32P-Postlabeling analysis is an ultrasensitive method for the detection and quantitation of DNA adducts and covalent modifications of the DNA. It consists of four main steps: (1) enzymatic digestion of DNA to 3′-monophosphate nucleosides; (2) enrichment of the adducts; (3) 5′OH-labeling of the adducts by T4 polynucleotide kinase-catalyzed transfer of 32P- orthophosphate from [γ-32P]ATP; and (4) chromatographic or electrophoretic separation of labeled adducts and detection and quantification by means of their radioactive decay. The assay requires only micrograms of DNA and is capable of detecting adduct levels as low as one adduct in 10 9-1010 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. © Springer Science+Business Media, New York 2013.