Abstract

³²P-postlabeling analysis is an ultrasensitive method for the detection and quantitation of carcinogen–DNA adducts. It consists of four principal steps: (i) enzymatic digestion of DNA to nucleoside 3'-monophosphates; (ii) enrichment of the adduct fraction of the DNA digest; (iii) 5'-labeling of the adducts by transfer of ³²P-orthophosphate from [-³²P]ATP mediated by polynucleotide kinase (PNK); (iv) chromatographic or electrophoretic separation of the labeled adducts or modified nucleotides and quantitation by measurement of their radioactive decay. The assay requires only microgram quantities of DNA and is capable of detecting adducts at frequencies as low as 1 in 10¹⁰ nt, making it applicable to the detection of events resulting from environmental exposures, or experiments using physiological concentrations of agents. It has a wide range of applications in human, animal and in vitro studies, and can be used for a wide variety of classes of compound and for the detection of adducts formed by complex mixtures. This protocol can be completed in 3 d.

1. Institute of Cancer Research, Brookes Lawley Building, Cotswold Road, Sutton, Surrey SM2 5NG, UK.

Correspondence to: David H Phillips¹ e-mail: david.phillips@icr.ac.uk

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