UNIVERSAL SYSTEM FOR DIRECTED NUCLEIC ACID CLEAVAGE

我们建议开发一种新的定向核酸切割系统。 这个系统是基于我们的发现，几个 耐热的DNA聚合酶可以被定向切割为特定的位点 核酸内切酶。该系统将具有耐热性、专属性、高效性和 易于自动化，并将在以下领域得到广泛应用 以核酸为基础的研究和诊断。在第一阶段，我们将寻求 克服了这项技术的最大限制，其 在切割长5‘臂方面效率低下。我们将研究以下方面的影响 影响α-氨基丁酸裂解效率的反应组成和条件 表征良好的衬底，执行全面的检查 不同盐类、二价阳离子、变性剂、缓冲液pH的影响 值、解旋酶和温度。我们还将使用突变技术来改变 并影响其与底物结构的相互作用。 在第二阶段，我们将优化所需的底物络合物形成 和核酸酶相互作用将这项技术发展成一种通用的， 单链和双链的定向核酸裂解系统 核酸。

We propose to develop a novel system for directed nucleic acid cleavage. This system is based upon our discovery that the 5' nucleases of several thermostable DNA polymerases can be directed to cleave as site-specific endonucleases. This system will be thermostable, specific, efficient and easily automatable, and will find broad application in the fields of nucleic acids-based research and diagnostics. In Phase I we will seek to overcome the most substantial limitation of this technology, its inefficiency at cleaving long 5' arms. We will examine the effects of reaction constituents and conditions on the efficiency of cleavage of a well-characterized substrate, performing a comprehensive examination of the effects of different salts, divalent cations, denaturants, buffer pH values, helicases and temperatures. We will also use mutagenesis to alter the protein and influence its interaction with the substrate structure. During Phase II we will optimize the required substrate complex formations and nuclease interactions to develop this technology into a generic, directed nucleic acid cleavage system for both single and double stranded nucleic acids.