Li Jishan, Chu Xia, Liu Yali, Jiang Jian-Hui, He Zhimin, Zhang Zhiwei, Shen Guoli, Yu Ru-Qin
State Key Laboratory for Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, PR China.
Nucleic Acids Res. 2005 Oct 27;33(19):e168. doi: 10.1093/nar/gni163.
The present study reported proof-of-principle for a genotyping assay approach that can detect single nucleotide polymorphisms (SNPs) through the gold nanoparticle assembly and the ligase reaction. By incorporating the high-fidelity DNA ligase (Tth DNA ligase) into the allele-specific ligation-based gold nanoparticle assembly, this assay provided a convenient yet powerful colorimetric detection that enabled a straightforward single-base discrimination without the need of precise temperature control. Additionally, the ligase reaction can be performed at a relatively high temperature, which offers the benefit for mitigating the non-specific assembly of gold nanoparticles induced by interfering DNA strands. The assay could be implemented via three steps: a hybridization reaction that allowed two gold nanoparticle-tagged probes to hybrid with the target DNA strand, a ligase reaction that generates the ligation between perfectly matched probes while no ligation occurred between mismatched ones and a thermal treatment at a relatively high temperature that discriminate the ligation of probes. When the reaction mixture was heated to denature the formed duplex, the purple color of the perfect-match solution would not revert to red, while the mismatch gave a red color as the assembled gold nanoparticles disparted. The present approach has been demonstrated with the identification of a single-base mutation in codon 12 of a K-ras oncogene that is of significant value for colorectal cancers diagnosis, and the wild-type and mutant type were successfully scored. To our knowledge, this was the first report concerning SNP detection based on the ligase reaction and the gold nanoparticle assembly. Owing to its ease of operation and high specificity, it was expected that the proposed procedure might hold great promise in practical clinical diagnosis of gene-mutant diseases.
本研究报告了一种基因分型检测方法的原理证明，该方法可通过金纳米颗粒组装和连接酶反应检测单核苷酸多态性（SNP）。通过将高保真DNA连接酶（Tth DNA连接酶）纳入基于等位基因特异性连接的金纳米颗粒组装中，该检测提供了一种方便而强大的比色检测方法，无需精确的温度控制就能直接进行单碱基区分。此外，连接酶反应可在相对较高的温度下进行，这有利于减轻干扰DNA链诱导的金纳米颗粒非特异性组装。该检测可通过三个步骤实施：杂交反应，使两个金纳米颗粒标记的探针与目标DNA链杂交；连接酶反应，在完全匹配的探针之间产生连接，而错配的探针之间不发生连接；在相对较高的温度下进行热处理，以区分探针的连接情况。当反应混合物加热使形成的双链变性时，完全匹配溶液的紫色不会恢复为红色，而错配的溶液则会因为组装的金纳米颗粒分散而呈现红色。本方法已通过鉴定K-ras癌基因第12密码子中的单碱基突变得到验证，这对结直肠癌诊断具有重要价值，并且成功区分了野生型和突变型。据我们所知，这是第一篇关于基于连接酶反应和金纳米颗粒组装的SNP检测的报告。由于其操作简便和高特异性，预计所提出的方法在基因突变异质性疾病的实际临床诊断中可能具有很大的前景。
