DEVELOPMENT OF AN IMPROVED MISMATCH BINDING PROTEIN

描述：(研究人员摘要)由于MutS在体内参与识别和修复DNA中的错配碱基，因此很可能成为需要在体外定量结合含有DNA的错配的应用的候选对象。然而，溶液中的MutS缺乏特异性，即不能区分错配的DNA分子和完全配对的DNA分子，以及相当弱的结合。固定化MutS具有很好的识别率，但识别能力较低。本项目的目的是从E.Coli MutS开始，利用诱变和强大的选择系统，开发一种改进的错配结合蛋白，能够在溶液中与含有错配或未配对碱基的DNA紧密结合。这样的蛋白质将在突变检测、克隆、基因发现和聚合酶链式反应保真度方面具有广泛的用途。拟议的商业应用：一种改进的错配结合蛋白将立即获得广泛的商业应用。它可用于目前的基因检查产品，包括错配结合珠和固定化错配结合蛋白平板，并将形成可用于突变检测和克隆应用的PCR清理试剂盒的基础。改进的错配结合蛋白也可能是通过下降基因发现方法进行身份识别的基础。

DESCRIPTION: (Investigator's abstract) MutS would appear, because of its involvement in vivo in the recognition and repair of mismatched bases in DNA, to be a likely candidate for applications requiring quantitative binding of mismatch containing DNA in vitro. However, MutS in solution suffers from lack of specificity, i.e., poor discrimination between mismatch containing and perfectly paired DNA molecules and from rather weak binding. Immobilized MutS has excellent discrimination, but low capacity. It is the aim of this project to start with E.Coli MutS and, by utilizing mutagenesis and a powerful selection system, develop an improved mismatch binding protein capable of extremely tight binding in solution to DNA containing mispaired or unpaired bases. Such a protein would have widespread utility in mutation detection, cloning, gene discovery and PCR fidelity. PROPOSED COMMERCIAL APPLICATION: An improved mismatch binding protein will have immediate and widespread commercial applications. It may be used in current Gene Check products including mismatch binding beads and immobilized mismatch binding protein plates and will form the basis of a PCR clean up kit, which can be used in both mutation detection and cloning applications. Improved mismatch binding protein may also be the basis of an identity by descent gene discovery method.