A Postlabeling Assay for Oxidative Damage
The 32P-postlabeling assay was originally devised by Randerath et al. (1981) to measure carcinogen-DNA adducts. In the procedure, DNA is first digested by micrococcal nuclease and calf spleen phosphodiesterase to give nucleoside 3′-monophosphates (normal and modified) that are subsequently labeled by incubation with [γ-32P]-ATP and T4 polynucleotide kinase. The radiolabeled compounds are then separated by two-dimensional TLC. The assay has two important advantages. First, there is no requirement for prelabeling the DNA, which makes the assay useful for the study of DNA lesions in tissues. Second, because of the availability of [γ-32P]-ATP of high specific activity, the assay permits detection at the femtomole level. There are, however, two major drawbacks:(1) the polynucleotide kinase must be able to act on the modified nucleoside 3′-monophosphate and (2) the resulting labeled modified nucleoside diphosphate must be separable from the high background of normal nucleoside diphosphates. These problems are well illustrated in the reports of efforts to detect thymine glycols, a well-known oxidative base lesion, in irradiated DNA (Reddy et al., 1991; Hegi et al., 1989). Furthermore, lesions that involve base loss, such as abasic sites and deoxyribose fragments, cannot be detected by this approach.
KeywordsAbasic Site Micrococcal Nuclease Modify Nucleoside Radioactive Band Buffer Chamber
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