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.
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.
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DOI：
10.1002/asia.200900661
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发表时间：
2010-05
期刊：
影响因子：
--
通讯作者：
Chen Song;Chen Zhang;Meiping Zhao
中科院分区：
文献类型：
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作者：
Chen Song;Chen Zhang;Meiping Zhao
DNA 3‘-磷酸酶在电离辐射或氧化应激等DNA损伤剂诱导的链断裂修复中发挥着独特的作用。本文介绍了一种快速筛选DNA 3‘-磷酸酶及其抑制剂的检测系统。设计了一种独特的模板底物与通用分子信标(U-MB)杂交，并在实时定量PCR中进行了检测过程。该方法成功地应用于两种典型的3‘-磷酸酶，即T4多核苷酸激酶磷酸酶(PNKP)和小牛肠道碱性磷酸酶(CIP)的活性和动力学监测。定量研究了肝素对T4PNKP的抑制作用和茶碱对CIP的抑制作用。该方法可用于各种3‘-磷酸酶及其抑制剂的灵敏、高通量和精确的测定。
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.