New approach to real-time nucleic acids detection: folding polymerase chain reaction amplicons into a secondary structure to improve cleavage of Förster resonance energy transfer probes in 5′-nuclease assays

塔克曼 放大器 聚合酶 生物 费斯特共振能量转移 核酸酶 分子信标 聚合酶 反聚合酶链反应 分子生物学 底漆二聚体 聚合酶链反应 核酸 聚合酶链反应优化 多重连接依赖探针扩增 实时聚合酶链反应 底漆(化妆品) DNA 遗传学 水热 荧光 多重聚合酶链反应 寡核苷酸 基因 化学 有机化学 物理 量子力学 外显子
作者
Igor V. Kutyavin
出处
期刊:Nucleic Acids Research [Oxford University Press]
卷期号:38 (5): e29-e29 被引量:20
标识
DOI:10.1093/nar/gkp1138
摘要

The article describes a new technology for real-time polymerase chain reaction (PCR) detection of nucleic acids. Similar to Taqman, this new method, named Snake, utilizes the 5'-nuclease activity of Thermus aquaticus (Taq) DNA polymerase that cleaves dual-labeled Förster resonance energy transfer (FRET) probes and generates a fluorescent signal during PCR. However, the mechanism of the probe cleavage in Snake is different. In this assay, PCR amplicons fold into stem-loop secondary structures. Hybridization of FRET probes to one of these structures leads to the formation of optimal substrates for the 5'-nuclease activity of Taq. The stem-loop structures in the Snake amplicons are introduced by the unique design of one of the PCR primers, which carries a special 5'-flap sequence. It was found that at a certain length of these 5'-flap sequences the folded Snake amplicons have very little, if any, effect on PCR yield but benefit many aspects of the detection process, particularly the signal productivity. Unlike Taqman, the Snake system favors the use of short FRET probes with improved fluorescence background. The head-to-head comparison study of Snake and Taqman revealed that these two technologies have more differences than similarities with respect to their responses to changes in PCR protocol, e.g. the variations in primer concentration, annealing time, PCR asymmetry. The optimal PCR protocol for Snake has been identified. The technology's real-time performance was compared to a number of conventional assays including Taqman, 3'-MGB-Taqman, Molecular Beacon and Scorpion primers. The test trial showed that Snake supersedes the conventional assays in the signal productivity and detection of sequence variations as small as single nucleotide polymorphisms. Due to the assay's cost-effectiveness and simplicity of design, the technology is anticipated to quickly replace all known conventional methods currently used for real-time nucleic acid detection.
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