Gening, L.V.; Volodin, A.A.; Kazachenko, K.Y.; Makarova, I.V.; Tarantul, V.Z. Estimation of the Mutagenic Potential of 8-Oxog in Nuclear Extracts of Mouse Cells Using the “Framed Mirror” Method. Methods Protoc.2020, 3, 3.
Gening, L.V.; Volodin, A.A.; Kazachenko, K.Y.; Makarova, I.V.; Tarantul, V.Z. Estimation of the Mutagenic Potential of 8-Oxog in Nuclear Extracts of Mouse Cells Using the “Framed Mirror” Method. Methods Protoc. 2020, 3, 3.
Gening, L.V.; Volodin, A.A.; Kazachenko, K.Y.; Makarova, I.V.; Tarantul, V.Z. Estimation of the Mutagenic Potential of 8-Oxog in Nuclear Extracts of Mouse Cells Using the “Framed Mirror” Method. Methods Protoc.2020, 3, 3.
Gening, L.V.; Volodin, A.A.; Kazachenko, K.Y.; Makarova, I.V.; Tarantul, V.Z. Estimation of the Mutagenic Potential of 8-Oxog in Nuclear Extracts of Mouse Cells Using the “Framed Mirror” Method. Methods Protoc. 2020, 3, 3.
Abstract
We propose an improved earlier described “mirror” method [1] for detecting in cell nuclear extracts mutations that arise in DNA during its replication due to misincorporation of deoxyadenosine-5’-monophosphate (dAMP) opposite 7,8-dihydro-8-oxoguanine (8-oxoG). The method is based on the synthesis of a complementary chain (“mirror”) by nuclear extracts of different mice organs on a template containing 8-oxoG inside and dideoxycytidine residue (ddC) at the 3’-end. The “mirror”was amplified by PCR using primers part of which was non-complementary to the template. It allowed obtaining the “framed mirror” products. The misincorporation of dAMP in “framed mirror” products forms an EcoRI restriction site. The restriction analysis of double-stranded “framed mirror” products allows a quantification of the mutation frequency in nuclear extracts. The data obtained showed that the mutagenic potential of 8-oxoG markedly varied in different organs of adult mice and embryos.
Keywords
DNA lesions; 7,8-dihydro-8-oxoguanine (8-oxoG); mutagenic activity; method of detection; cell nuclear extracts; mice organs and embryos
Subject
Biology and Life Sciences, Biochemistry and Molecular Biology
Copyright:
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