Sequencing of DNA Lesions Facilitated by Site-Specific Excision via Base Excision Repair DNA Glycosylases Yielding Ligatable Gaps.
Sequencing of DNA Lesions Facilitated by Site-Specific Excision via Base Excision Repair DNA Glycosylases Yielding Ligatable Gaps.
复制标题
DOI：
10.1021/jacs.5b11563
复制
发表时间：
2016-01-20
影响因子：
15
通讯作者：
Burrows CJ
中科院分区：
文献类型：
--
作者：
Riedl J;Fleming AM;Burrows CJ
基因组中核苷酸的修饰可能导致突变或参与基因表达的调节，因此，找到修饰位点是一个有价值的目标。除了胞嘧啶的表观遗传标记之外，尚未开发出对商业测序仪上的修饰位点进行测序的稳健方法。在此，开发了一种对 DNA 修饰位点进行测序的方法，该方法利用碱基切除修复途径中发现的 DNA 糖基化酶来切除修饰。这种方法在修饰位点处产生一个缺口，该缺口被 T4-DNA 连接酶密封，从而产生缺少修饰的产物链。测序后，修饰的核苷酸被报告为缺失突变，从而确定了其位置。该方法用于检测合成寡脱氧核苷酸中 KRAS 基因密码子 12 中的尿嘧啶 (U) 或 8-oxo-7,8-二氢鸟嘌呤 (OG)。此外，将 OG 修饰位点置于质粒中的 VEGF 启动子中并进行测序。该方法仅需要市售材料，并且可以在任何测序平台上付诸实践，使得该方法在发现 DNA 修饰方面具有广泛的潜力。
Modifications to nucleotides in the genome can lead to mutations or are involved in regulation of gene expression, and therefore, finding the site of modification is a worthy goal. Robust methods for sequencing modification sites on commercial sequencers have not been developed beyond the epigenetic marks on cytosine. Herein, a method to sequence DNA modification sites was developed that utilizes DNA glycosylases found in the base excision repair pathway to excise the modification. This approach yields a gap at the modification site that is sealed by T4-DNA ligase yielding a product strand missing the modification. Upon sequencing, the modified nucleotide is reported as a deletion mutation, identifying its location. This approach was used to detect a uracil (U) or 8-oxo-7,8-dihydroguanine (OG) in codon 12 of the KRAS gene in synthetic oligodeoxynucleotides. Additionally, an OG modification site was placed in the VEGF promoter in a plasmid and sequenced. This method requires only commercially available materials and can be put into practice on any sequencing platform, allowing this method to have broad potential for finding modifications in DNA.