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. 1994 Jul 19;91(15):7232–7236. doi: 10.1073/pnas.91.15.7232

A 32P-postlabeling method for detecting unstable N-7-substituted deoxyguanosine adducts in DNA.

D Yu 1, T Q Niu 1, P Austin-Ritchie 1, D B Ludlum 1
PMCID: PMC44373  PMID: 8041772

Abstract

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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Selected References

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