Estimation of the Mutagenic Potential of 8-Oxog in Nuclear Extracts of Mouse Cells Using the “Framed Mirror” Method
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 meth...
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MDPI AG
2020-01-01
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| Series: | Methods and Protocols |
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| Online Access: | https://www.mdpi.com/2409-9279/3/1/3 |
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| author | Leonid V. Gening Alexandr A. Volodin Konstantin Y. Kazachenko Irina V. Makarova Vyacheslav Z. Tarantul |
| author_facet | Leonid V. Gening Alexandr A. Volodin Konstantin Y. Kazachenko Irina V. Makarova Vyacheslav Z. Tarantul |
| author_sort | Leonid V. Gening |
| collection | DOAJ |
| description | 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 <i>EcoRI</i> 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. |
| first_indexed | 2024-12-13T10:42:46Z |
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| id | doaj.art-923f0f11e512478f80f71b0e2067c8a1 |
| institution | Directory Open Access Journal |
| issn | 2409-9279 |
| language | English |
| last_indexed | 2024-12-13T10:42:46Z |
| publishDate | 2020-01-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Methods and Protocols |
| spelling | doaj.art-923f0f11e512478f80f71b0e2067c8a12022-12-21T23:50:25ZengMDPI AGMethods and Protocols2409-92792020-01-0131310.3390/mps3010003mps3010003Estimation of the Mutagenic Potential of 8-Oxog in Nuclear Extracts of Mouse Cells Using the “Framed Mirror” MethodLeonid V. Gening0Alexandr A. Volodin1Konstantin Y. Kazachenko2Irina V. Makarova3Vyacheslav Z. Tarantul4Institute of Molecular Genetics, Kurchatov Sq. 2, Moscow 123182, RussiaInstitute of Molecular Genetics, Kurchatov Sq. 2, Moscow 123182, RussiaInstitute of Molecular Genetics, Kurchatov Sq. 2, Moscow 123182, RussiaInstitute of Molecular Genetics, Kurchatov Sq. 2, Moscow 123182, RussiaInstitute of Molecular Genetics, Kurchatov Sq. 2, Moscow 123182, RussiaWe 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 <i>EcoRI</i> 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.https://www.mdpi.com/2409-9279/3/1/3dna lesions7,8-dihydro-8-oxoguanine (8-oxog)mutagenic activitymethod of detectioncell nuclear extractsmice organs and embryos |
| spellingShingle | Leonid V. Gening Alexandr A. Volodin Konstantin Y. Kazachenko Irina V. Makarova Vyacheslav Z. Tarantul Estimation of the Mutagenic Potential of 8-Oxog in Nuclear Extracts of Mouse Cells Using the “Framed Mirror” Method Methods and Protocols dna lesions 7,8-dihydro-8-oxoguanine (8-oxog) mutagenic activity method of detection cell nuclear extracts mice organs and embryos |
| title | Estimation of the Mutagenic Potential of 8-Oxog in Nuclear Extracts of Mouse Cells Using the “Framed Mirror” Method |
| title_full | Estimation of the Mutagenic Potential of 8-Oxog in Nuclear Extracts of Mouse Cells Using the “Framed Mirror” Method |
| title_fullStr | Estimation of the Mutagenic Potential of 8-Oxog in Nuclear Extracts of Mouse Cells Using the “Framed Mirror” Method |
| title_full_unstemmed | Estimation of the Mutagenic Potential of 8-Oxog in Nuclear Extracts of Mouse Cells Using the “Framed Mirror” Method |
| title_short | Estimation of the Mutagenic Potential of 8-Oxog in Nuclear Extracts of Mouse Cells Using the “Framed Mirror” Method |
| title_sort | estimation of the mutagenic potential of 8 oxog in nuclear extracts of mouse cells using the framed mirror method |
| topic | dna lesions 7,8-dihydro-8-oxoguanine (8-oxog) mutagenic activity method of detection cell nuclear extracts mice organs and embryos |
| url | https://www.mdpi.com/2409-9279/3/1/3 |
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