A 32P-postlabeling method for detecting unstable N-7-substituted deoxyguanosine adducts in DNA
Department of Pharmacology and Molecular Toxicology
Animals; Cattle; Chromatography, High Pressure Liquid; DNA; DNA Damage; Deoxyguanosine; Genetic Techniques; Phosphorus Radioisotopes
Life Sciences | Medicine and Health Sciences
Many antitumor agents, including the mustards, form N-7 deoxyguanosine adducts in DNA that are difficult to quantitate by the 32P-postlabeling procedure because of their instability. We have developed a method that is successful for the analysis of such adducts using, as a prototype mustard, 14C-labeled bis(2-chloroethyl)sulfide. This agent forms the unstable product 7-hydroxyethylthioethyldeoxyguanosine in DNA. By performing enzymatic digestions to 3'-deoxynucleotides at 10 degrees C, including a second N-7-substituted guanine deoxynucleotide as an internal standard, removing most of the unmodified nucleotides and [32P]ATP on disposable anion columns, and measuring the labeled products after separation on a C18 column, we are able to detect 1 unstable N-7 deoxyguanosine adduct in 10(7) normal nucleotides with good precision.
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Citation: Proc Natl Acad Sci U S A. 1994 Jul 19;91(15):7232-6.
Proceedings of the National Academy of Sciences of the United States of America
Yu, Dong; Niu, Tian-Qi; Austin-Ritchie, Paula; and Ludlum, David B., "A 32P-postlabeling method for detecting unstable N-7-substituted deoxyguanosine adducts in DNA" (1994). Open Access Articles. 1808.