DEVELOPMENT OF A SYSTEM FOR HIGH-SPEED GENE SYNTHESIS

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”物质的困难。传统的重组克隆昂贵、耗时且范围有限。我们建议利用大规模的寡核苷酸合成来解决这一需求，并将开发一种高通量基因合成器，一种能够将计算机中设计的DNA或蛋白质序列转化为用于蛋白质表达的长DNA分子的仪器。一个原型系统包括：1)在计算机上设计、编辑、数据库和修改基因和载体序列；2)生成一组覆盖整个设计的重叠寡核苷酸序列；3)编程自动合成寡核苷酸集；4)机器人组装组件寡核苷酸并将其连接成大的DNA链；5)将得到的DNA转化到大肠杆菌中进行基因复制和蛋白质表达。该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的质粒。利用这种方法，可以生产出大多数或所有的人类蛋白质，作为重组细菌阵列，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；2)一种高通量基因和蛋白质生产技术，可能导致用于功能和结构分析的人类蛋白质阵列的商业应用。

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.