Working Paper Article Version 1 This version is not peer-reviewed

Estimation of The Mutagenic Potential of 8-oxoG in Nuclear Extracts of Mouse Cells Using The “Framed Mirror” Method

Version 1 : Received: 12 November 2019 / Approved: 14 November 2019 / Online: 14 November 2019 (09:00:19 CET)

How to cite: 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. Preprints 2019, 2019110153 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. Preprints 2019, 2019110153

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.

Subject Areas

DNA lesions; 7,8-dihydro-8-oxoguanine (8-oxoG); mutagenic activity; method of detection; cell nuclear extracts; mice organs and embryos

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