A 32P-postlabeling method for detecting unstable N-7-substituted deoxyguanosine adducts in DNA.
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.
Yu
D
D
Department of Pharmacology, University of Massachusetts Medical School, Worcester 01655.
Niu
T Q
TQ
Austin-Ritchie
P
P
Ludlum
D B
DB
D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000737:chemistry*; D004249:DNA Damage; D003849:Deoxyguanosine / Q000032:analysis*; D005821:Genetic Techniques; D010761:Phosphorus Radioisotopes
D010761:Phosphorus Radioisotopes; D004247:DNA; D003849:Deoxyguanosine