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

Abstract

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 AluΙ, EcoRΙ, XhoΙ, and SacΙ at smoothly controlled temperature. Cleavage efficiencies were determined, and the potential applications of this method were discussed. Furthermore, the AluΙ and EcoRΙ 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.