Functional Selection of High Performance Enzymes

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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.