Schmeiser Heinz H, Stiborova Marie, Arlt Volker M
Research Group Genetic Alterations in Carcinogenesis, German Cancer Research Center-DKFZ, Heidelberg, Germany.
Methods Mol Biol. 2013;1044:389-401. doi: 10.1007/978-1-62703-529-3_21.
³²P-Postlabeling analysis is an ultrasensitive method for the detection and quantitation of DNA adducts and covalent modifications of the DNA. It consists of four main steps: (1) enzymatic digestion of DNA to 3'-monophosphate nucleosides; (2) enrichment of the adducts; (3) 5'OH-labeling of the adducts by T4 polynucleotide kinase-catalyzed transfer of ³²P-orthophosphate from [γ-³²P]ATP; and (4) chromatographic or electrophoretic separation of labeled adducts and detection and quantification by means of their radioactive decay. The assay requires only micrograms of DNA and is capable of detecting adduct levels as low as one adduct in 10⁹-10¹⁰ normal nucleotides. It is applicable to a wide range of investigations in human, animal, and in vitro studies including monitoring exposure to environmental or occupational carcinogens, determining whether a chemical or a complex mixture has genotoxic properties, elucidation of the toxicological pathways of carcinogens, and monitoring DNA repair.
³²P后标记分析是一种用于检测和定量DNA加合物及DNA共价修饰的超灵敏方法。它包括四个主要步骤：（1）将DNA酶解为3'-单磷酸核苷；（2）加合物的富集；（3）通过T4多核苷酸激酶催化从[γ-³²P]ATP转移³²P-正磷酸盐对加合物进行5'OH标记；（4）对标记的加合物进行色谱或电泳分离，并通过其放射性衰变进行检测和定量。该检测方法仅需微克级的DNA，能够检测低至10⁹-10¹⁰个正常核苷酸中一个加合物的加合物水平。它适用于人类、动物和体外研究中的广泛调查，包括监测对环境或职业致癌物的暴露、确定一种化学物质或复杂混合物是否具有遗传毒性、阐明致癌物的毒理学途径以及监测DNA修复。
