Gening Leonid V, Shevchenko Oleg V, Kazachenko Konstantin Y, Tarantul Vyacheslav Z
Institute of Molecular Genetics, Kurchatov Sq. 2, Moscow 123182, Russia.
Methods Protoc. 2018 Aug 27;1(3):32. doi: 10.3390/mps1030032.
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 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.
我们提出了一种改进的方法，用于检测由于脱氧腺苷-5'-单磷酸（dAMP）与7,8-二氢-8-氧代鸟嘌呤（8-oxoG）错配而在DNA中产生的突变。该方法基于使用含有8-oxoG的模板合成互补链（“镜像”产物）。dAMP在“镜像”产物中的错配形成了位点。通过对“镜像”产物进行PCR获得的双链DNA的限制性分析允许对诱变频率进行定量。此外，对单链“镜像”产物的单链构象多态性（SSCP）分析表明，不同的DNA聚合酶在8-oxoG对面仅掺入dA或dC。开发该技术所采用的拟议方法也可应用于其他损伤的研究，包括单个损伤和聚集损伤。
