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Development of a high-throughput screening platform for DNA 3'-phosphatases and their inhibitors based on a universal molecular beacon and quantitative real-time PCR.

DNA 3'-phosphatases play a unique role in the repair of strand breaks induced by DNA damaging agents, such as ionizing radiation or oxidative stress. In this paper, we present an efficient detection system for rapid screening of DNA 3'-phosphatases and their inhibitors. A unique template substrate has been designed to hybridize with the universal molecular beacon (U-MB), and the detection process is carried out in a quantitative real-time PCR. The method is successfully applied to monitor the activity and kinetics of two typical 3'-phosphatases, that is, T4 polynucleotide kinase phosphatase (PNKP) and calf intestinal alkaline phosphatase (CIP). The inhibition effect of heparin on T4 PNKP and theophylline on CIP is also quantitatively characterized. The proposed method is demonstrated to be very useful for sensitive, high-throughput, and precise measurement of various 3'-phosphatases and their inhibitors.