Functional Selection of High Performance Enzymes

描述（由申请人提供）：微流体系统正在成为用于基于细胞的高通量分析的至关重要的工具，用于包括基因分型、测序、基因表达分析、核酸扩增和诊断在内的应用。芯片实验室设备通常被设计为在连续流中执行微化学反应，需要外部组件，例如用于处理溶液的微型阀和微型泵，以及控制一切的复杂仪器。这些系统中的大多数是大型的，它们的制造是昂贵的，并且它们需要专有的化学品和一次性用品，这增加了它们的成本。该提案的一个目标是开发一种负担得起的简单工具，用于使用基于液滴的微流体技术将来自单个细胞的基因组和蛋白质组数据相关联。这种简单的手持设备不需要电连接或仪器，但将允许形成精确控制的液滴，使生物反应器的大规模平行阵列适合于酶筛选，定向进化，大DNA片段的乳液PCR，单细胞高通量筛选和许多其他应用。这将为不适合这些任务的传统化学和更复杂和昂贵的新一代仪器提供替代品。该提议的第二个目标是利用该装置通过其自动扩增含有其基因的大操纵子的能力来从头发现新的热稳定病毒复制体。我们将通过定向进化来优化这些多亚基酶，以进行具有细胞复制样保真度的100 kb PCR，从而能够对高度复杂的疾病位点进行简单的遗传分析。 公共卫生相关性：生物学中的许多复杂问题在没有大规模操纵和处理的情况下基本上是难以解决的。大型复杂疾病基因座的遗传分析极其昂贵和耗时。我们建议开发一种简单的系统，可以轻松生产数百万个微型生物反应器来研究细胞，蛋白质和基因。我们将利用这个装置来开发强大的新酶，用于扩增非常大的DNA。这项工作将使研究人员能够更好地了解正常和疾病情况下的细胞，蛋白质和基因。

DESCRIPTION (provided by applicant): Microfluidic systems are emerging as critically important tools for high throughput cell based analysis for applications including genotyping, sequencing, gene expression analysis, nucleic acid amplification, and diagnostics. Lab-on-a-chip devices that are conventionally designed to perform microchemical reactions in a continuous flow require external components, such as microvalves, and micropumps for handling solution, and complicated instruments to control everything. Most of these systems are large, their fabrication is expensive and they require proprietary chemistry and disposables, which increases their costs. One goal of this proposal is to develop an affordable, simple tool for correlating genomic and proteomic data from individual cells using droplet based microfluidic technology. This simple handheld device requires no electrical connections or instrumentation, but will allow for the formation of precisely controlled droplets, enabling massively parallel arrays of bioreactors suitable for enzyme screening, directed evolution, emulsion PCR of large DNA fragments, single-cell high throughput screening and numerous other applications. This will provide an alternative to conventional chemistries that are inadequate for these tasks and more complicated and expensive newer-generation instruments. The second goal of this proposal is to utilize this device to discovery novel thermostable viral replisomes de novo by their ability to auto amplify large operons containing their genes. We will optimize these multi-subunit enzymes through directed evolution to perform 100 kb PCR with cellular replication-like fidelity, enabling straightforward genetic analysis of highly complex disease loci. PUBLIC HEALTH RELEVANCE: Many complex problems in biology are essentially intractable without large-scale manipulation and processing. The genetic analysis of large complex disease loci is prohibitively expensive and time consuming. We propose to develop a simple system to readily produce millions of micro bioreactors to study cells, proteins and genes. We will use this device to develop powerful new enzymes for amplifying very large DNAs. This work will enable researchers to better understand cells, proteins and genes in normal and disease situations.