High-throughput ultra-sensitive discrimination of single nucleotide polymorphism via click chemical ligation
High-throughput ultra-sensitive discrimination of single nucleotide polymorphism via click chemical ligation
复制标题
通过点击化学连接高通量超灵敏区分单核苷酸多态性
DOI：
10.1039/c9an01672d
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发表时间：
2020
期刊：
影响因子：
4.2
通讯作者：
Zhang Xin-Xiang
中科院分区：
文献类型：
--
作者：
Zhou Qian-Yu;Zhong Xin-Ying;Zhao Ling-Li;Wang Li-Juan;Zhou Ying-Lin;Zhang Xin-Xiang
单核苷酸多态(SNPs)已被证明是疾病诊断、预后和疾病发病机制的重要生物标志物。在这里，利用自组装寡核苷酸夹心结构和强大的化学反应的优势，我们开发了一种简单、高通量和有效的比色分析技术，称为基于CuAAC的连接辅助分析(CuAAC-LA)，用于DNA-BIND 96孔板上的SNP检测。通过在两个寡核苷酸探针上标记5‘-叠氮基和3’-炔基，靶DNA可以直接进行铜(I)催化的炔-叠氮环加成(CuAAC)点击反应。由于单核苷酸错配引起的双链稳定性的微小差异被这些反应基团对不稳定双链的连接反应的空间效应放大，CuAAC-LA在存在大量野生型靶标的情况下表现出对突变类型靶标的超灵敏识别能力。低至0.05%的SNP检测结果优于以往报道的各种DNA连接酶方法，表明DNA模板直接点击反应是一种简单快速的合成方法，有望取代连接酶用于SNP检测。
Single nucleotide polymorphisms (SNPs) have been proven to be important biomarkers for disease diagnosis, prognosis and disease pathogenesis. Here, taking the advantages of a self-assembled oligonucleotide sandwich structure and robust chemical reactions, we have developed a simple, high-throughput and effective colorimetric analytical technique termed CuAAC-based ligation-assisted assays (CuAAC-LA) for SNP detection using a DNA-BIND 96-well plate. With the 5′-azide and 3′-alkyne groups labelled on two oligonucleotide probes, the target DNA can direct a Cu(I)-catalyzed alkyne–azide cycloaddition (CuAAC) click reaction. Since the small difference in duplex stability caused by a single-nucleotide mismatch was amplified by the steric effects of these reactive groups for the ligation reaction of an unstable duplex, CuAAC-LA exhibited an ultra-sensitive discrimination ability for a mutant type target in the presence of large amounts of wild type targets. As low as 0.05% SNP could be clearly detected, which was better than most previously reported methods by various DNA ligases, indicating that a simple and rapid synthetic method i.e., the DNA template-directed click reaction held the potential to replace the ligase for SNP detection.