"Mirror" Method to Estimate Mutagenic Activity of DNA Lesions

We propose an improved method for detecting mutations that arise in DNA due to misincorporations of deoxyadenosine-5'-monophosphate (dAMP) opposite 7,8-dihydro-8-oxoguanine (8-oxoG). The method is based on the synthesis of complementary chains ("mirror" products) using a template containing 8-oxoG. The misincorporation of dAMP in the "mirror" product forms EcoRI sites. The restriction analysis of double-stranded DNAs obtained by PCR of "mirror" product allows quantification of the mutagenesis frequency. In addition, single-strand conformational polymorphism (SSCP) analysis of the single-stranded "mirror" products shows that different DNA polymerases only incorporate dA or dC opposite 8-oxoG. The proposed approach used in developing this technique can be applied in the study of other lesions as well, both single and clustered.