A Digital Microfluidic Systems for Gene Synthesis, Sequencing and Recovery

用于基因合成、测序和恢复的数字微流控系统

• 批准号: 8532939
• 负责人: Mark D ADAMS
• 金额: $ 22.02万
• 依托单位: J. CRAIG VENTER INSTITUTE, INC.
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-01 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8532939
• 关键词: Aliquot; Automation; Base Pairing; Base Sequence; Biomedical Research; Blood capillaries; Bypass; Chemistry; Cloning; Coupling; DNA; DNA Sequence; DNA amplification; DNA biosynthesis; Development; Dideoxy Chain Termination DNA Sequencing; Enzymes; Escherichia coli; Genes; Genome; Goals; In Vitro; Institutes; Length; Liquid substance; Logic; Methods; Microfluidics; Oligonucleotides; Phase; Process; Protein Engineering; Protocols documentation; Reaction; Reading; Recovery; Solid; Synthesis Chemistry; System; Technology; Time; Work; base; capillary; chemical synthesis; cost; design; digital; flexibility; gene synthesis; improved; innovation; microchip; next generation sequencing; parallel processing; processing speed; pyrosequencing; single molecule; synthetic biology; synthetic construct; synthetic protein; tool

DESCRIPTION (provided by applicant): The design and in vitro synthesis of genes or long DNA molecules is an important and powerful tool in biomedical research, synthetic biology and protein engineering. While the actual synthesis and assembly reactions are highly amenable to automation and parallel processing, the major bottlenecks for making in vitro DNA synthesis a routine, high throughput and high speed process are the verification and recovery of the full length and error free synthetic DNA molecules which rely on laborious E. coli cloning and Sanger sequencing technologies. We propose the development of a bench top microfluidic system that has the ability to perform DNA synthesis, sequence verification and recovery of the desired full length DNA molecules. The system incorporates the innovative and flexible digital microfluidic platform developed by Advanced Liquid Logic, Inc. (ALL) with an efficient gene assembly protocol developed by the J. Craig Venter Institute (JCVI). The resulting system will employ pyrosequencing chemistry on a digital microfluidic cartridge to enable sequence verification and recovery of full length, error- free DNA molecules bypassing the laborious cloning and capillary based sequencing processes. The system will also enable direct coupling of the microchip-based oligonucleotide synthesis platform to the downstream assembly, sequencing and recovery process, further reducing the costs and improving the overall efficiency of gene and genome synthesis.

描述（由申请人提供）：基因或长DNA分子的设计和体外合成是生物医学研究、合成生物学和蛋白质工程中重要而有力的工具。虽然实际的合成和组装反应非常适合于自动化和并行处理，但使体外DNA合成成为常规、高通量和高速过程的主要瓶颈是全长和无错误合成DNA分子的验证和回收，这依赖于费力的E. coli克隆和桑格测序技术。我们建议开发一种台式微流体系统，该系统能够进行DNA合成、序列验证和回收所需的全长DNA分子。该系统采用了由Advanced Liquid Logic，Inc.开发的创新和灵活的数字微流体平台。(ALL)利用由J.克雷格文特尔研究所（JCVI）开发的高效基因组装方案。所得到的系统将在数字微流体盒上采用焦磷酸测序化学，以使得能够绕过费力的克隆和基于毛细管的测序过程进行全长、无错误DNA分子的序列验证和回收。该系统还将实现基于微芯片的寡核苷酸合成平台与下游组装、测序和回收过程的直接耦合，进一步降低成本并提高基因和基因组合成的整体效率。