A 32P-postlabeling method for detecting unstable N-7-substituted deoxyguanosine adducts in DNA
UMass Chan AffiliationsDepartment of Pharmacology and Molecular Toxicology
Chromatography, High Pressure Liquid
Medicine and Health Sciences
MetadataShow full item record
AbstractMany 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.
Proc Natl Acad Sci U S A. 1994 Jul 19;91(15):7232-6.