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题名: Continuous monitoring of restriction endonuclease cleavage activity by universal molecular beacon light quenching coupled with real-time polymerase chain reaction
作者: Li, Xiaomin1;  Song, Chen1;  Zhao, Meiping1;  Li, Yuanzong1
关键词: universal molecular beacon ;  real-time polymerase chain reaction ;  restriction endonucleases
刊名: ANALYTICAL BIOCHEMISTRY
发表日期: 2008-10-01
DOI: 10.1016/j.ab.2008.06.027
卷: 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
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.
关键词[WOS]: ECO-RI ENDONUCLEASE ;  DNA CLEAVAGE ;  RECOGNITION SITE ;  SYNTHETIC OLIGODEOXYNUCLEOTIDES ;  FLUOROMETRIC ASSAY ;  SEQUENCES FLANKING ;  FLUORESCENCE ;  KINETICS ;  MICROCHIP
语种: 英语
WOS记录号: WOS:000258729700001
Citation statistics: 
内容类型: 期刊论文
URI标识: http://cas-ir.dicp.ac.cn/handle/321008/141064
Appears in Collections:中国科学院大连化学物理研究所_期刊论文

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作者单位: 1.Peking Univ, BNLMS, Key Lab Bioorgan Chem & Mol Engn, Coll Chem & Mol Engn, Beijing 100871, Peoples R China

Recommended Citation:
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.
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