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The 32P-postlabeling assay for DNA adducts

Nature Protocols volume 2, pages 2772–2781 (2007)Cite this article

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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 [γ-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.

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Figure 1: Methods of 32P-postlabeling.
Figure 2: Diagram showing multidirectional thin-layer chromatography procedures for the resolution of 32P-labeled adducts on polyethyleneimine-cellulose.
Figure 3: Examples of thin-layer chromatograms of 32P-postlabeled DNA.
Figure 4: Safety precautions.
Figure 5

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  • 17 January 2008

    In Figure 1 of the version of this article originally published, the arrow between “*pXp” and “*pX” should not have been bold, as it does not indicate a procedure for which a protocol is given in the article. In addition, the legend should have contained, as the third sentence, the following information: “For procedures leading to *pX as the final products, see Table 2, column 3.” The errors have been corrected in the HTML and PDF versions of the article.

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    David H Phillips & Volker M Arlt

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Phillips, D., Arlt, V. The 32P-postlabeling assay for DNA adducts. Nat Protoc 2, 2772–2781 (2007). https://doi.org/10.1038/nprot.2007.394

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