32P-Postlabeling Analysis of DNA Adducts
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32P-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 DNA sample; enrichment of the adducts; radiolabeling of the adducts by T4 kinase-catalyzed transference of 32P-orthophosphate from [γ-32P]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 109–1010 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.
Key wordsDNA adducts 32P-postlabeling T4 polynucleotide kinase Nuclease P1 Complex mixtures DNA repair Oxidative damage to DNA Carcinogens Mutagens Genotoxicity Environmental carcinogens Chemotherapeutic drugs HPLC DNA digestions
- 1.Phillips DH (2002) The formation of DNA adducts. In: Allison MR (ed) The Cancer Handbook. Macmillan, London, pp 293–306Google Scholar
- 11.Pfau W, Lecoq S, Hughes NC, Seidel A, Platt KL, Grover PL, Phillips DH (1993) Separation of 32P-labelled 3′,5′-bisphosphate adducts by HPLC. In: Phillips DH, Castegnaro M, Bartsch H (eds) Postlabelling Methods for Detection of DNA Adducts. IARC, Lyon, pp 233–242Google Scholar
- 18.Phillips DH, Castegnaro M (1993) Results of an interlaboratory trial of 32P-postlabelling. In: Phillips DH, Castegnaro M, Bartsch H (eds) Postlabelling Methods for Detection of DNA Damage. IARC, Lyon, pp 35–49Google Scholar