Continuous monitoring of restriction endonuclease cleavage activity by   universal molecular beacon light quenching coupled with real-time   polymerase chain reaction

doi:10.1016/j.ab.2008.06.027

CORC > 北京大学 > 化学与分子工程学院

	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
刊名	分析生物化学
	2008
关键词	universal molecular beacon real-time polymerase chain reaction restriction endonucleases ECO-RI ENDONUCLEASE DNA CLEAVAGE RECOGNITION SITE SYNTHETIC OLIGODEOXYNUCLEOTIDES FLUOROMETRIC ASSAY SEQUENCES FLANKING FLUORESCENCE KINETICS MICROCHIP
DOI	10.1016/j.ab.2008.06.027
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.; http://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcApp=PARTNER_APP&SrcAuth=LinksAMR&KeyUT=WOS:000258729700001&DestLinkType=FullRecord&DestApp=ALL_WOS&UsrCustomerID=8e1609b174ce4e31116a60747a720701 ; Biochemical Research Methods; Biochemistry & Molecular Biology; Chemistry, Analytical; SCI(E); PubMed; 12; ARTICLE; 1; 1-7; 381
语种	英语
内容类型	期刊论文
源URL	[http://ir.pku.edu.cn/handle/20.500.11897/150209]
专题	化学与分子工程学院
推荐引用方式 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]. 分析生物化学,2008.
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.分析生物化学.
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".分析生物化学 (2008).