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32P-postlabelling for the Sensitive Detection of DNA Adducts

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Genetic Toxicology

Part of the book series: Methods in Molecular Biology ((MIMB,volume 817))

Abstract

32P-postlabelling is a technique originally described by Kurt Randerath and colleagues for the sensitive detection of damage produced in DNA by reactive chemicals or genotoxins. The procedure essentially entails the enzymatic digestion of DNA to nucleoside 3′-monophosphates which are then radioactively labelled using T4 polynucleotide kinase and [γ32P]-adenosine triphosphate. Adducted nucleoside-3′-5′-bisphosphates are then separated from their normal counterparts by thin layer chromatography. Prior to the development of the assay, quantification of DNA adducts was confined to studies that utilised compounds synthesised to be isotopically labelled with tritium or carbon-14. As such, these studies were limited to specific and recognised genotoxins that could be administered only in the laboratory to cultures or animals. With 32P-postlabelling it was possible not only to determine DNA adduct induction by a relatively uncharacterised suspected carcinogen, but also following exposure to complex mixtures containing a multitude of known and unknown potential genotoxins. The small amount of DNA required to perform the 32P-postlabelling assay also meant that human biomonitoring studies using readily obtainable tissues, such as lymphocytes, were possible. Using the standard 32P-postlabelling method, it is possible to detect a single DNA adduct in 107 to 108 normal nucleotides. The subsequent development of several enhancement methods improved this detection rate to one adduct in 1010 nucleotides. For these reasons, the 32-postlabelling assay represents an extremely versatile and extremely sensitive method to detect and monitor DNA damage.

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Authors and Affiliations

  1. Department of Biochemistry and Cell Biology, Institute of Integrative Biology, University of Liverpool, Biosciences Building, Crown Street, Liverpool, L69 7ZB, UK

    Nigel J. Jones

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Correspondence to Nigel J. Jones .

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Editors and Affiliations

  1. Cedarmount 7, Lyndhurst, SO43 7ED, United Kingdom

    James M. Parry

  2. Cedarmount 7, Lyndhurst, SO43 7ED, Hampshire, United Kingdom

    Elizabeth M. Parry

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Jones, N.J. (2012). 32P-postlabelling for the Sensitive Detection of DNA Adducts. In: Parry, J., Parry, E. (eds) Genetic Toxicology. Methods in Molecular Biology, vol 817. Springer, New York, NY. https://doi.org/10.1007/978-1-61779-421-6_10

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  • DOI: https://doi.org/10.1007/978-1-61779-421-6_10

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