ISOLATION OF A THERMOPHILIC REPLICASE

嗜热复制酶的分离

• 批准号: 6019160
• 负责人: Nebojsa JANJIC
• 金额: $ 35.96万
• 依托单位: ENZYCO, INC.
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-09-30 至 2001-08-01
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6019160
• 关键词: DNA binding protein; DNA directed DNA polymerase; chimeric proteins; method development; nucleic acid reconstitution; polymerase chain reaction; protein purification; replicase; structural genes; thermophilic organism; transfection /expression vector

In our phase I studies, we isolated a subassembly of a T. thermophilus DNA polymerase III holoenzyme, establishing the existence of a standard eubacterial replicase in extreme thermophiles. The objectives of our proposed phase II studies are to develop methods for obtaining large quantities of the Tth DNA polymerase III holoenzyme and, exploiting the enormous catalytic efficiency and processivity of this enzyme, to develop very long PCR techniques that enable routine amplification of 100 kb or longer sequences. Such an improvement in this important method will enhance basic molecular biology research and genome research by extending the size of the steps taken when walking along the genome. The availability of a suitable enzyme for very long PCR will enhance diagnostic procedures for cancer, pathogens and genetic defects and will greatly extend the technology for positional cloning and physical marker linkage analysis. PROPOSED COMMERCIAL APPLICATION: The development of a megabase processivity thermophilic replicase should have a dramatic impact on the 100. million dollar/year thermophilic polymerae market.

在我们的第一阶段研究中，我们分离出了T的一个亚组。嗜热 DNA聚合酶III全酶，建立存在的标准 极端嗜热菌中的真细菌复制酶。 我们的目标 拟议的第二阶段研究是开发获得大规模 大量的Tth DNA聚合酶III全酶， 这种酶巨大的催化效率和持续合成能力， 开发非常长的PCR技术，使常规扩增 100 kb或更长的序列。 这一重要领域的进步 该方法将加强基础分子生物学研究和基因组研究 通过扩展当沿着基因组沿着行走时所采取的步骤的大小。 对于非常长的PCR来说，合适的酶的可用性将提高 癌症、病原体和遗传缺陷的诊断程序， 大大扩展了定位克隆和物理标记技术 连锁分析 拟定商业应用： 一个巨大的嗜热复制酶的发展， 对100强产生了巨大的影响。百万美元/年 聚合物市场。