滚动圆复制
哑铃
环介导等温扩增
DNA
核酸
底漆(化妆品)
方向性
杂交探针
互补序列
序列(生物学)
化学
等温过程
生物物理学
核酸热力学
纳米技术
生物系统
生物
分子生物学
基序列
材料科学
生物化学
物理
DNA复制
数学
有机化学
统计
热力学
生理学
作者
Yi Long,Xiaoming Zhou,Da Xing
标识
DOI:10.1016/j.bios.2013.02.003
摘要
Sequence-specific nucleic acid detection is playing a more and more important role in modern life sciences. Traditional rolling circle amplification (RCA) involves multiple distinct reaction steps and the experiment result is influenced by multiple factors. What's more, a main limitation of traditional RCA is that each target strand hybridizes with only one padlock probe, and this 1:1 hybridization ratio limits the sensitivity. Here we have proposed target sequence recycled rolling circle amplification (TR-RCA) to increase sensitivity by one step. We demonstrated that our method can not only make RCA occur, but also one target DNA can be reused and thus achieving self-recycle. In TR-RCA, the dumbbell probe recognizes the target DNA and hybridizes with it, and then the stem of the dumbbell probe is opened, after that the opened area anneals with the primer and triggers RCA. At the same time, after a target is displaced, it recognizes and hybridizes with another dumbbell probe, triggering the next cycle of RCA. This amplification method is achievable at a constant temperature simply by mixing dumbbell probes, target DNA, primers, and other chemical complexes together in one tube. Our method has significant advantages in ease of operation. And the results indicate that the target DNA can be detected at fM level with high specificity.
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