MUTANTS OF TAQ DNA POLYMERASE

描述(改编自调查人员的摘要)：本提案基于 假设热启动对高效和 大多数聚合酶链式反应的可靠扩增，特别是要求最高的 聚合酶链式反应。热开关可以通过以下任一种方式添加到PCR协议中 手动或内置方法，所有这些方法都有诸如狭窄等缺点 条件、交叉污染和/或缺乏对长时间聚合酶链式反应的适用性。这个 研究人员建议开发一种Taq聚合酶突变体 由于将引入对冷敏感的突变，因此可热切换。 建议开发一种突变的Taq DNA聚合酶，其 在室温(反应装置)下比野生型活跃，但正常情况下 活跃在65-70度，但仍能抵抗95度。调查人员 将对突变基因进行测序(预计每个基因都有一个以上的突变 从突变中)，并测试单个突变以确定哪些突变 对冷敏感。最后，他们将提纯突变酶和组合突变酶， 看看高要求的聚合酶链式反应(短的和长的)是否需要热启动 通过在广泛的条件下使用它们而得到改进。 建议的商业应用：不可用。

DESCRIPTION (Adapted from the investigator's abstract): This proposal is based on the premise that a hot start is beneficial or necessary for efficient and reliable amplification of most PCR reactions, particularly the most demanding PCR reactions. A heat switch can be added to PCR protocols by any of several manual or built-in methods, all of which have drawbacks such as narrow conditions, cross-contamination, and/or lack of applicability to long PCR. The investigators propose development of a Taq polymerase mutant that is heat-switchable by virtue of cold-sensitive mutations that will be introduced. Proposed is the development of a mutant Taq DNA polymerase that is much less active than the wild-type at room (reaction setup) temperature, yet normally active at 65-70 degrees, and still resistant to 95 degrees. The investigators will sequence the mutant genes (expected to harbor more than one mutation each from the mutagenesis) and test single mutations to identify ones that confer cold-sensitivity. Finally they will purify mutant and combined-mutant enzymes, to see if demanding PCR (short and long) that requires a hot start can be improved by their use under a broad range of conditions. PROPOSED COMMERCIAL APPLICATION: Not available.