Key Laboratory of Medicinal Chemistry and Molecular Diagnosis of the Ministry of Education, Key Laboratory of Analytical Science and Technology of Hebei Province (Project Number: 22567620H), State Key Laboratory of New Pharmaceutical Preparations and Excipients, College of Chemistry and Materials Science, Hebei University, Baoding 071002, China.
Key Laboratory of Applied Surface and Colloid Chemistry, Ministry of Education; Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry & Chemical Engineering, Shaanxi Normal University, Xi'an 710119, China.
Anal Chem. 2024 Aug 13;96(32):13285-13290. doi: 10.1021/acs.analchem.4c02621. Epub 2024 Jul 30.
Accurate detection of site-specific 5-hydroxymethylcytosine (5hmC) in genomic DNA is of great significance, but it is technically challenging to directly distinguish very low levels of 5hmC from their abundant cytosine/5-methylcytosine (C/5mC) analogues. Herein, we wish to propose a selective ligase-mediated mechanism (SLim) that can directly discriminate 5hmC from C/5mC with a high specificity without the use of any sample processing protocol. In this new design, we discovered that HiFi Taq DNA Ligase can well tolerate the mismatched 5hmC/A base-pairing and then effectively ligate the associated nicking site while the mismatched 5mC/A or C/A pairs cannot be recognized by HiFi Taq DNA Ligase, providing a new way for direct and selective discriminating 5hmC from its similar analogues. Ultrasensitive and selective quantification of site-specific 5hmC is realized by coupling the SLim with polymerase chain reaction (PCR) or loop-mediated isothermal amplification (LAMP).
准确检测基因组 DNA 中的特定 5-羟甲基胞嘧啶（5hmC）具有重要意义，但直接区分非常低水平的 5hmC 与其丰富的胞嘧啶/5-甲基胞嘧啶（C/5mC）类似物在技术上具有挑战性。在此，我们提出了一种选择性连接酶介导的机制（SLim），该机制可以在不使用任何样品处理方案的情况下，以高特异性直接区分 5hmC 与 C/5mC。在这个新设计中，我们发现 HiFi Taq DNA 连接酶可以很好地容忍错配的 5hmC/A 碱基对，然后有效地连接相关的缺口位点，而错配的 5mC/A 或 C/A 对不能被 HiFi Taq DNA 连接酶识别，为直接和选择性区分 5hmC 与其类似物提供了一种新方法。通过将 SLim 与聚合酶链反应（PCR）或环介导等温扩增（LAMP）偶联，实现了对特定 5hmC 的超灵敏和选择性定量。
