Fleming Aaron M, Ding Yun, Burrows Cynthia J
University of Utah, Salt Lake City, UT, United States.
University of Utah, Salt Lake City, UT, United States.
Methods Enzymol. 2017;591:187-210. doi: 10.1016/bs.mie.2017.03.004. Epub 2017 Apr 7.
The DNA base guanine (G) can be oxidatively modified to 8-oxo-7,8-dihydroguanine (OG). Extraction of genomic DNA followed by nuclease digestion and mass spectrometry analysis has found OG is present at background levels of ~1 out of 10 Gs; however, this approach cannot determine the locations for the OGs in the genome. Thus, in this methods report, we outline three different methods (A, B, and C) for sequencing OG in DNA. Method A sequences OG by utilizing the base excision repair pathway to delete the OG nucleotide from the DNA that is then detected by Sanger sequencing as a deletion signature. Method B sequences OG by harnessing the base excision repair pathway to convert OG to an unnatural DNA base pair followed by Sanger sequencing to locate the unnatural base pair indicating where OG was located. Method C (i.e., OG-Seq) takes genomic DNA sheared to ~150bps followed by selectively biotinylating the OG-containing fragments for affinity purification and enrichment of the OG-modified strands. The OG-modified fragments are sequenced on a next-generation sequencing platform to locate OG on the genomic scale with a resolution of ~150bps. The methods outlined are then compared and contrasted allowing researchers to select the one that best suits their experimental goals.
DNA碱基鸟嘌呤（G）可被氧化修饰为8-氧代-7,8-二氢鸟嘌呤（OG）。提取基因组DNA，随后进行核酸酶消化和质谱分析，结果发现OG以大约每10个G中有1个的背景水平存在；然而，这种方法无法确定基因组中OG的位置。因此，在本方法报告中，我们概述了三种不同的DNA中OG测序方法（方法A、方法B和方法C）。方法A利用碱基切除修复途径对DNA中的OG核苷酸进行删除，然后通过桑格测序将其作为缺失特征进行检测，从而对OG进行测序。方法B利用碱基切除修复途径将OG转化为非天然DNA碱基对，随后通过桑格测序定位该非天然碱基对，以此指示OG的位置，从而对OG进行测序。方法C（即OG-Seq）将基因组DNA剪切成约150bp片段，然后选择性地对含OG的片段进行生物素化，用于亲和纯化和富集OG修饰的链。将OG修饰的片段在新一代测序平台上进行测序，以在基因组规模上定位OG，分辨率约为150bp。然后对所概述的方法进行比较和对比，以便研究人员选择最适合其实验目标的方法。
