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(32)P-postlabeling analysis of DNA adducts

Research output: Chapter in Book/Report/Conference proceedingChapter

Original languageEnglish
Title of host publicationMolecular Toxicology Protocols
EditorsPhouthone Keohavong, Stephen G Grant
Place of PublicationTotowa, N.J.
PublisherHumana Press
Pages127-138
Number of pages12
Edition2nd
ISBN (Print)9781627037389
DOIs
Published2014

Publication series

NameMethods in Molecular Biology
PublisherHumana Press
Volume1105
ISSN (Print)1064-3745

King's Authors

Abstract

(32)P-Postlabeling analysis is an ultra-sensitive method for the detection of DNA adducts, such as those formed directly by the covalent binding of carcinogens and mutagens to bases in DNA and other DNA lesions resulting from modification of bases by endogenous or exogenous agents (e.g., oxidative damage). The procedure involves four main steps: enzymatic digestion of the DNA sample; enrichment of the adducts; radiolabeling of the adducts by T4 kinase-catalyzed transference of (32)P-orthophosphate from [γ-(32)P]ATP; chromatographic separation of labeled adducts; and detection and quantification by means of their radioactive decay. Using 10 μg of DNA or less, it is capable of detecting adduct levels as low as 1 adduct in 10(9)-10(10) normal nucleotides. It is applicable to a wide range of investigations, including monitoring human exposure to environmental or occupational carcinogens, determining whether a chemical has genotoxic properties, analysis of the genotoxicity of complex mixtures, elucidation of the pathways of activation of carcinogens, and monitoring DNA repair.

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