| 高灵敏靶标自环化滚环扩增检测的动态接头构建 |
| |
| 引用本文： | 王晨儒,安然,梁兴国.高灵敏靶标自环化滚环扩增检测的动态接头构建[J].中国海洋大学学报(自然科学版),2021(5). |
| |
| 作者姓名： | 王晨儒 安然 梁兴国 |
| |
| 作者单位： | 中国海洋大学食品科学与工程学院;青岛海洋科学与技术试点国家实验室海洋药物与生物制品功能实验室 |
| |
| 基金项目： | 国家重点研究发展计划项目(2019YFD0901701,2019YFC1604603);山东省自然科学基金项目(ZR2019BC096)资助。 |
| |
| 摘 要： | 针对靶标自环化滚环扩增(TC-RCA)难以高灵敏度检测的难题,本文探究了扩增过程中靶标环化效率低的原因,并构建了新型动态接头以提高靶标DNA的环化灵敏度。通过采用含有发卡结构的动态接头(18 nt),使接头的两端产生连接活性差异,探讨发卡接头打开与闭合的动态平衡在提高环化灵敏度中的作用。具体研究了发卡动态接头的稳定性和磷酸化对连接及环化效率的影响。研究表明,增加接头两端的连接活性差异可提高环化灵敏度(106 copies/μL),从而揭示了TC-RCA灵敏度低的根本原因。由于许多过量的接头同时连接到靶标DNA两端后,靶标无法环化,造成仅有少部分靶标环化为RCA模板,导致极低浓度的靶标无法发生RCA。在此基础上,采用另一种10 nt的短链动态接头,使检测灵敏度提高了两个数量级(达104 copies/μL)。本研究为TC-RCA中接头的设计提供了新思路,对双链DNA环化机理的深入研究具有重要意义。
|
| 关 键 词： | 滚环扩增 发卡结构 DNA环化 核酸检测 接头 |
Construction of Dynamic Adaptors for Improving Detection Efficiency of RCA Based on Target Circularization |
| |
| WANG Chen-Ru,AN Ran,LIANG Xing-Guo.Construction of Dynamic Adaptors for Improving Detection Efficiency of RCA Based on Target Circularization[J].Periodical of Ocean University of China,2021(5). |
| |
| Authors: | WANG Chen-Ru AN Ran LIANG Xing-Guo |
| |
| Institution: | (College of Food Science and Engineering, Ocean University of China, Qingdao 266003, China;Functional Laboratory of Marine Drugs and Biological Products, Pilot National Laboratory of Marine Science and Technology(Qingdao), Qingdao 266237, China) |
| |
| Abstract: | To solve the problem of low sensitivity in target circularization for rolling circle amplification(TC-RCA),in this study,we explored the reasons for low efficiency of target circularization,and constructed new dynamic adaptors to improve the circularization sensitivity.Firstly,dynamic adaptors with hairpin structure(18 nt)were designed to make the ligation activity different between the two ends of adaptors,and the influence of dynamic equilibrium between opening and closing of the hairpin on the circularization sensitivity was discussed.The effect of the stability and phosphorylation of the hairpin adaptors on ligation efficiency was studied in detail.The results showed that increasing the difference of ligation activity at both ends of adaptors improves the circularization sensitivity(106 copies/μL),indicating the reason for low sensitivity for TC-RCA.Because a large number of dsDNA adaptors are simultaneously ligated to both ends of target DNA,the target cannot be circulated.As a result,only a small number of targets are circulated into RCA templates,thus reducing the detection sensitivity.Moreover,the detection sensitivity was increased by another 10-nt short dynamic adaptor(104 copies/μL or less).Our findings in this study are also significant to improve the efficiency of double-stranded DNA circula-rization in genetic engineering. |
| |
| Keywords: | rolling circle amplification hairpin structure DNA circularization detecting of nucleic acid adaptors |
| 本文献已被 CNKI 维普 等数据库收录！ |
|
