New approach to real-time nucleic acids detection: folding polymerase chain reaction amplicons into a secondary structure to improve cleavage of Forster resonance energy transfer probes in 5'-nuclease assays.
New approach to real-time nucleic acids detection: folding polymerase chain reaction amplicons into a secondary structure to improve cleavage of Forster resonance energy transfer probes in 5'-nuclease assays.
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DOI：
10.1093/nar/gkp1138
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发表时间：
2010-03
影响因子：
14.9
通讯作者：
Kutyavin IV
中科院分区：
文献类型：
--
作者：
Kutyavin IV
本文介绍了一种新的实时聚合酶链反应（PCR）检测核酸的技术。与Taqman类似，这种名为Snake的新方法利用了水生栖热菌（Taq）DNA聚合酶的5′-核酸酶活性，该聚合酶可切割双标记的Förster共振能量转移（FRET）探针，并在PCR过程中产生荧光信号。然而，Snake中探针切割的机制是不同的。在该测定中，PCR扩增子折叠成茎环二级结构。FRET探针与这些结构之一的杂交导致Taq的5′-核酸酶活性的最佳底物的形成。Snake扩增子中的茎环结构是通过一个PCR引物的独特设计引入的，该引物携带一个特殊的5′-flap序列。结果发现，在这些5′-瓣序列的一定长度处，折叠的Snake扩增子对PCR产率的影响非常小，如果有的话，但有利于检测过程的许多方面，特别是信号产生率。与Taqman不同，Snake系统倾向于使用具有改善的荧光背景的短FRET探针。Snake和Taqman的头对头比较研究表明，这两种技术在对PCR方案变化（例如引物浓度、退火时间、PCR不对称性的变化）的反应方面差异大于相似之处。已确定了Snake的最佳PCR方案。将该技术的实时性能与许多常规测定进行了比较，包括Taqman，3′-MGB-Taqman，Molecular Beacon和Scorpion引物。试验表明，Snake在信号产生和检测小至单核苷酸多态性的序列变异方面取代了传统的测定方法。由于该检测的成本效益和设计简单，该技术预计将迅速取代目前用于实时核酸检测的所有已知常规方法。
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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DOI：
10.1073/pnas.85.20.7652
发表时间：
1988-10-01
影响因子：
11.1
作者：
GYLLENSTEN, UB;ERLICH, HA
通讯作者：
ERLICH, HA
影响因子：
2.9
作者：
LADUCA, RJ;FAY, PJ;BAMBARA, RA
通讯作者：
BAMBARA, RA
DOI：
10.1073/pnas.85.23.8790
发表时间：
1988-12-01
影响因子：
11.1
作者：
CARDULLO, RA;AGRAWAL, S;WOLF, DE
通讯作者：
WOLF, DE
影响因子：
2.9
作者：
Harvey, JJ;Lee, SP;Han, MK
通讯作者：
Han, MK
影响因子：
46.9
作者：
Lyamichev, V;Mast, AL;Brow, MAD
通讯作者：
Brow, MAD