32P-Postlabeling for Detection of DNA Adducts
It is generally believed that the formation of DNA adducts by covalent interaction of electrophilic species of carcinogens (ultimate carcinogens) with macromolecules, particularly DNA, is an essential first step in the multistage process of carcinogenesis (Miller and Miller, 1981). Until the early 1980s, use of radiolabeled carcinogens was the main method to determine the binding of chemical carcinogens to DNA. Since then several methods have been utilized for nonradio-labeled carcinogens. The assays that are practiced currently are based on specific antibodies, fluorescence properties of adducts, gas chromatography/mass spectrometry, and 32P-postlabeling. These assays require a few micrograms to several hundred micrograms of DNA, with a detection limit of 1 adduct per 106 to 1010 nucleotides, depending on the method (Beach and Gupta, 1992). The 32P-postlabeling assay has emerged as the major tool for measuring DNA adducts because of its ultrasensitivity and applicability to theoretically any DNA-damaging agent, irrespective of its chemical nature, including unknowns. Over 100 individual agents or complex mixtures have been tested in rodents and aquatic systems in vivo and rodent and human cells in vitro (Beach and Gupta, 1992).
KeywordsAmmonium Hydroxide Ammonium Formate Adduct Level Micrococcal Nuclease Major Spot
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