Continuous monitoring of restriction endonuclease cleavage activity by universal molecular beacon light quenching coupled with real-time polymerase chain reaction
Continuous monitoring of restriction endonuclease cleavage activity by universal molecular beacon light quenching coupled with real-time polymerase chain reaction
复制标题
通过通用分子信标光猝灭结合实时聚合酶链式反应连续监测限制性内切酶裂解活性
DOI：
10.1016/j.ab.2008.06.027
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发表时间：
2008-10-01
影响因子：
2.9
通讯作者：
Li, Yuanzong
中科院分区：
文献类型：
--
作者：
Li, Xiaomin;Song, Chen;Li, Yuanzong
我们描述了一种通过使用通用分子信标（U-MB）结合实时聚合酶链反应（PCR）灵敏监测限制性内切酶动力学和活性的方法。该方法用于监测密封反应管中DNA切割的进程，并提供更准确和高通量的检测。模板具有与U-MB杂交的通用尾，并且剩余序列与限制性内切酶消化产物之一互补。U-MB被消化产物的延伸取代，荧光猝灭。利用这一概念，一种通用的荧光探针可以用于不同的酶分析系统。在本文所述的工作中，在平稳控制的温度下，用限制性内切酶EcoRI、XhoI和SacI进行均相测定。测定了裂解效率，并讨论了该方法的潜在应用。此外，在分子水平上在不同底物浓度下在线监测EcoRI和EcoRI切割反应，并计算K-m、V-max和K-cat值。结果表明，基于实时PCR的限制性内切酶测定的U-MB监测将是非常有用的高通量，灵敏，精确的酶活性筛选和进化生物技术分析的测定。(c)2008年爱思唯尔公司All rights reserved.
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.