DEVELOPMENT OF A SYSTEM FOR HIGH-SPEED GENE SYNTHESIS

高速基因合成系统的开发

• 批准号: 6144490
• 负责人: GLEN A EVANS
• 金额: $ 9.81万
• 依托单位: EGEA BIOSCIENCES, INC.
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-04-01 至 2000-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6144490
• 关键词: Escherichia coli; biomedical equipment development; biotechnology; computer program /software; computer system design /evaluation; gene expression; high throughput technology; molecular biology information system; nucleic acid chemical synthesis; oligonucleotides; plasmids; transfection /expression vector

The Human Genome Sequencing Project is generating a vast amount of raw sequence information. However, a limitation in the analysis of new human proteins is the difficulty of converting digital sequence information into "wet" material in the laboratory. Conventional recombinant cloning is expensive, time consuming and limited in scope. We propose utilizing large scale oligonucleotide synthesis to address this need and will develop a high-throughput gene synthesizer, an instrument capable of converting a DNA or protein sequence designed in the computer into long DNA molecules for protein expression. A prototype system consists of: 1) designing, editing, databasing, and modifying gene and vector sequences in silico; 2) generating a set of overlapping oligonucleotide sequences covering the entire design; 3) programming automated synthesis of the oligonucleotide set; 4) robotically assembling the component oligonucleotides and ligating these into large DNA strands; and 5) transforming the resulting DNA into E. coli for gene replication and protein expression. The prototype system is capable of producing plasmids expressing recombinant human proteins at one gene/instrument/day and acceptable cost. Using this approach it is conceivable to produce most or all human proteins as arrays of recombinant bacteria for further functional analysis. PROPOSED COMMERCIAL APPLICATIONS: This work will result in 1) a prototype instrument for gene and genome synthesis that could be the basis of a commercial product; and 2) a technology for high throughput gene and protein production that could result in commercial applications of human protein arrays for functional and structural analysis.

人类基因组测序计划正在产生大量的原始序列信息。然而，分析新的人类蛋白质的一个限制是在实验室中将数字序列信息转换为“湿”材料的困难。常规的重组克隆是昂贵的、耗时的并且范围有限。我们建议利用大规模的寡核苷酸合成来满足这一需求，并将开发一种高通量基因合成仪，一种能够将计算机中设计的DNA或蛋白质序列转化为长DNA分子用于蛋白质表达的仪器。原型系统包括：1）通过计算机设计、编辑、数据库和修饰基因和载体序列; 2）生成一组覆盖整个设计的重叠寡核苷酸序列; 3）编程寡核苷酸组的自动合成; 4）机器人组装组成寡核苷酸并将其连接成大DNA链;和5）将所得DNA转化为大肠杆菌。coli中进行基因复制和蛋白表达。原型系统能够以一个基因/仪器/天和可接受的成本生产表达重组人蛋白质的质粒。使用这种方法，可以想象产生大部分或全部人类蛋白质作为重组细菌的阵列用于进一步的功能分析。拟议的商业应用：这项工作将导致1）基因和基因组合成的原型仪器，可能是商业产品的基础;和2）高通量基因和蛋白质生产的技术，可能导致人类蛋白质阵列的功能和结构分析的商业应用。