Phillips David H, Arlt Volker M
Department of Analytical, Environmental & Forensic Sciences, MRC-PHE Centre for Environmental & Health, King's College London, London, UK.
Methods Mol Biol. 2020;2102:291-302. doi: 10.1007/978-1-0716-0223-2_16.
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 DNA sample; enrichment of the adducts; radiolabeling of the adducts by T4 kinase-catalyzed transference of P-orthophosphate from [γ-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 10-10 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.
后标记分析是一种超灵敏的检测DNA加合物的方法，这些加合物例如是由致癌物和诱变剂与DNA中的碱基共价结合直接形成的，以及由内源性或外源性试剂对碱基进行修饰所产生的其他DNA损伤（例如氧化损伤）。该过程包括四个主要步骤：DNA样品的酶促消化；加合物的富集；通过T4激酶催化从[γ-P]ATP转移正磷酸对加合物进行放射性标记；标记加合物的色谱分离，以及通过其放射性衰变进行检测和定量。使用10μg或更少的DNA，它能够检测低至每10-10个正常核苷酸中有1个加合物的加合物水平。它适用于广泛的研究，包括监测人类对环境或职业致癌物的暴露、确定一种化学物质是否具有遗传毒性、分析复杂混合物的遗传毒性、阐明致癌物的活化途径以及监测DNA修复。
