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 one of the major contributors to DNA damage and mutagenesis. In this study, we developed a modification of allele-specific PCR to detect CC-->TT mutations caused by oxidative damage. These tandem mutations have been previously demonstrated to be indicative of oxygen damage in the absence of UV-irradiation. Using a CC target site in the rat DNA polymerase beta (pol beta) gene and a thermostable restriction enzyme that cuts the wild type sequence but not the TT mutation, we demonstrate that the TT mutation can be preferentially amplified from plasmid DNA damaged by oxygen radicals but not other DNA-damaging agents. We evaluated the potential utility of this assay in screening for mutations in cells and in analyzing those that arise during clonal proliferation in carcinogenesis.
Newcomb
T G
TG
Department of Pathology, The Joseph Gottstein Memorial Cancer Research Laboratory, University of Washington, Seattle, WA 98195, USA.
Allen
K J
KJ
Tkeshelashvili
L
L
Loeb
L A
LA
D004249:DNA Damage; D004252:DNA Mutational Analysis; D019951:DNA Polymerase beta / Q000235:genetics; D017931:DNA Primers; D005609:Free Radicals / Q000633:toxicity*; D006367:HeLa Cells; D006801:Humans; D006861:Hydrogen Peroxide / Q000633:toxicity; D009154:Mutation / Q000235:genetics*; D010957:Plasmids / Q000187:drug effects; D016133:Polymerase Chain Reaction / Q000379:methods*; D017382:Reactive Oxygen Species
D017931:DNA Primers; D005609:Free Radicals; D017382:Reactive Oxygen Species; D006861:Hydrogen Peroxide; D019951:DNA Polymerase beta