Detection of tandem CC -> TT mutations induced by oxygen radicals using mutation-specific PCR

DNA lesions caused by reactive oxygen species (ROS) are considered to be on e of the major contributors to DNA damage and mutagenesis. In this study, w e developed a modification of allele-specific PCR to detect CC -->TT mutati ons caused by oxidative damage. These tandem mutations have been previously demonstrated to be indicative of oxygen damage in the absence of UV-irradi ation. Using a CC target site in the rat DNA polymerase beta (pol beta) gen e and a thermostable restriction enzyme that cuts the wild type sequence bu t not the TT mutation, we demonstrate that the TT mutation can be preferent ially amplified from plasmid DNA damaged by oxygen radicals but not other D NA-damaging agents. We evaluated the potential utility of this assay in scr eening for mutations in cells and in analyzing those that arise during clon al proliferation in carcinogenesis. (C) 1999 Elsevier Science B.V. All righ ts reserved.