Continuous monitoring of restriction endonuclease cleavage activity by universal molecular beacon light quenching coupled with real-time polymerase chain reaction
文献类型：期刊论文
| 作者 | Li, Xiaomin; Song, Chen; Zhao, Meiping; Li, Yuanzong |
| 刊名 | ANALYTICAL BIOCHEMISTRY
 |
| 出版日期 | 2008-10-01 |
| 卷号 | 381期号:1页码:1-7 |
| 关键词 | Universal Molecular Beacon Real-time Polymerase Chain Reaction Restriction Endonucleases |
| ISSN号 | 0003-2697 |
| 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. |
| 语种 | 英语 |
| WOS记录号 | WOS:000258729700001 |
| 出版者 | ACADEMIC PRESS INC ELSEVIER SCIENCE |
| 源URL | [http://ir.iccas.ac.cn/handle/121111/64763]  |
| 专题 | 中国科学院化学研究所 |
| 通讯作者 | Zhao, Meiping |
| 作者单位 | Peking Univ, BNLMS, Key Lab Bioorgan Chem & Mol Engn, Coll Chem & Mol Engn, Beijing 100871, Peoples R China |
| 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]. 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. |
入库方式： OAI收割
来源：化学研究所
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