通过DNA聚合剪切放大体系提高RNA的检测灵敏度

PDF全文下载：2014030265 

王明印， 任锐*

 （青岛科技大学 化学与分子工程学院，山东 青岛 266042）

 摘要： 基于循环的DNA剪切循环放大分子机器构建了一个RNA传感器。该分子机器以RNA为输入，产生大量的DNA片段，并替换报告探针上的荧光DNA从而产生荧光信号，实现对靶RNA浓度的放大检测。本分子机器分为两部分，反应部分和报告部分。在反应部分，以靶RNA为输入条件，以一个特殊设计的探针为反应模板引发一个自发连续的DNA聚合剪切反应网络，重复产生大量信号DNA链；这些信号DNA链进入报告部分，通过杂交替换反应从一个报告探针上替换下带有荧光DNA序列，释放到溶液中。这样通过剪切产生的大量DNA适体序列被释放到溶液中，并替换报告探针上的荧光DNA，实现信号的放大。

 关键词： RNA检测;分子机器； 剪切； DNA聚合

 中图分类号： R 392文献标志码： A

  收稿日期： 20130329

基金项目： 国家自然科学基金项目(21025523)；国家重点基础研究发展计划（973计划）项目(2010CB732404)；长江学者与创新团队计划项目(PCSIRT).

作者简介： 王明印(1988—)，男，硕士研究生.*通信联系人.

 Detection Sensitivity of RNA Improved by DNA Polymerase Shear Amplification System

 WANG Mingyin, REN Rui

 (CollegeofChemistryand Molecular Engineering,QingdaoUniversityof Science and Technology,Qingdao266042,China)

 Abstract: An RNA sensor was constructed based on a cycling DNA molecular machine that coupled DNA polymerization and restricted nicking. This molecular machine took RNA strand as the input, and produced reporting RNA strands with much amplified concentration (compared to the inputted RNA strand), which replaced the fluorescence signal strand in a fluorescence probe to produce fluorescence signal. This system was involved the cycling of the DNA polymerization and nicking, forming a cycling system. In this amplification system, a probe with two strands was used, with one strand for the production of the signal DNA strand, while the other for the recognition of the target RNA and the cycling of the DNA polymerization and restricted nicking. These two reactions were repeated due to the cycling of the target RNA strand on the second strand; and the cycling increased the throughput of the molecular machine, thus bringing forth even more amplification. In this system, the optimal condition for the DNA polymerizationRNA transcription was explored.

 Key words: RNA detection; molecular machines; shear; DNA polymerization