Estimation of the Mutagenic Potential of 8-Oxog in Nuclear Extracts of Mouse Cells Using the "Framed Mirror" Method

• Published in: Methods and protocols Vol. 3; no. 1; p. 3
• Main Authors: Gening, Leonid V, Volodin, Alexandr A, Kazachenko, Konstantin Y, Makarova, Irina V, Tarantul, Vyacheslav Z
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 03-01-2020; MDPI
• Subjects: 7,8-dihydro-8-oxoguanine (8-oxog); Adenosine monophosphate; cell nuclear extracts; dna lesions; DNA replication; Gene mutation; Health aspects; method of detection; mice organs and embryos; mutagenic activity; United States; cell nuclear extracts; 7,8-dihydro-8-oxoguanine (8-oxoG); mice organs and embryos; DNA lesions; method of detection; mutagenic activity
• ISSN: 2409-9279; 2409-9279
• DOI: 10.3390/mps3010003

We propose an improved earlier described "mirror" method for detecting in cell nuclear extracts mutations that arise in DNA during its replication due to the misincorporation of deoxyadenosine-5'-monophosphate (dAMP) opposite 7,8-dihydro-8-oxoguanine (8-oxoG). This method is based on the synthesis of a complementary chain ("mirror") by nuclear extracts of different mice organs on a template containing 8-oxoG 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
restriction site. The restriction analysis of double-stranded "framed mirror" products allows a quantification of the mutation frequency in nuclear extracts. The data obtained show that the mutagenic potential of 8-oxoG markedly varied in different organs of adult mice and embryos.