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Continuous monitoring of restriction endonuclease cleavage activity by universal molecular beacon light quenching coupled with real-time polymerase chain reaction
Li, Xiaomin; Song, Chen; Zhao, Meiping; Li, Yuanzong
关键词Universal Molecular Beacon Real-time Polymerase Chain Reaction Restriction Endonucleases
刊名ANALYTICAL BIOCHEMISTRY
2008-10-01
DOI10.1016/j.ab.2008.06.027
381期:1页:1-7
收录类别SCI
文章类型Article
WOS标题词Science & Technology ; Life Sciences & Biomedicine ; Physical Sciences
类目[WOS]Biochemical Research Methods ; Biochemistry & Molecular Biology ; Chemistry, Analytical
研究领域[WOS]Biochemistry & Molecular Biology ; Chemistry
关键词[WOS]ECO-RI ENDONUCLEASE ; DNA CLEAVAGE ; RECOGNITION SITE ; SYNTHETIC OLIGODEOXYNUCLEOTIDES ; FLUOROMETRIC ASSAY ; SEQUENCES FLANKING ; FLUORESCENCE ; KINETICS ; MICROCHIP
英文摘要We describe a method for sensitive monitoring of restriction endonuclease kinetics and activity by use of a universal molecular beacon (U-MB) coupled with real-time polymerase chain reaction (PCR). The method is used to monitor the progress of DNA cleavage in a sealed reaction tube and offers more accurate and high-throughput detection. The template has a universal tail hybridized with the U-MB and the remaining sequence is complementary to one of the restriction endonuclease digestion products. The U-MB is replaced by the extension of digested product and the fluorescence quenches. With this concept, one universal fluorescence probe can be used in different enzyme analytical systems. In the work described here, homogenous assays were performed with the restriction endonucleases AluI, EcoRI, XhoI, and Sacl at smoothly controlled temperature. Cleavage efficiencies were determined, and the potential applications of this method were discussed. Furthermore, the AluI and EcoRI cleavage reactions were monitored online at varying substrate concentrations at the molecular level, and K(m), V(max), and K(cat) Values were calculated. The results suggest that U-MB monitoring of restriction endonuclease assays based on real-time PCR will be very useful for high-throughput, sensitive, and precise assays for enzyme activity screening and evolutionary biotechnology analysis. (c) 2008 Elsevier Inc. All rights reserved.
语种英语
WOS记录号WOS:000258729700001
引用统计
文献类型期刊论文
条目标识符http://cas-ir.dicp.ac.cn/handle/321008/141064
专题中国科学院大连化学物理研究所
作者单位Peking Univ, BNLMS, Key Lab Bioorgan Chem & Mol Engn, Coll Chem & Mol Engn, Beijing 100871, Peoples R China
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GB/T 7714
Li, Xiaomin,Song, Chen,Zhao, Meiping,et al. Continuous monitoring of restriction endonuclease cleavage activity by universal molecular beacon light quenching coupled with real-time polymerase chain reaction[J]. ANALYTICAL BIOCHEMISTRY,2008,381(1):1-7.
APA Li, Xiaomin,Song, Chen,Zhao, Meiping,&Li, Yuanzong.(2008).Continuous monitoring of restriction endonuclease cleavage activity by universal molecular beacon light quenching coupled with real-time polymerase chain reaction.ANALYTICAL BIOCHEMISTRY,381(1),1-7.
MLA Li, Xiaomin,et al."Continuous monitoring of restriction endonuclease cleavage activity by universal molecular beacon light quenching coupled with real-time polymerase chain reaction".ANALYTICAL BIOCHEMISTRY 381.1(2008):1-7.
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