Efficient Simultaneous Synthesis of Large Arrays of Oligonucleotides

描述(申请人提供)：第一阶段是开发一种大阵列寡核苷酸合成仪器，它将极大地降低成本并简化巨型碱基大小的双链DNA片段的构建。与传统的DNA合成器相比，这项新技术使用的合成试剂不到1/40，每次反应将产生100至500纳克范围的寡聚。这一过程能够合成多达10,000个重叠寡核苷酸，这些寡核苷酸可以通过退火和连接组装成双链DNA。这项技术结合了传统的寡核苷酸合成化学和新的光刻阀门技术，将合成试剂输送到大量单独的反应室。在初步数据中，该系统的一个原型成功地进行了测试。该装置使用一系列计算机生成的阀垫来实现小型寡聚合成反应室的开启或关闭。该原型允许以每碱基0.012美元的试剂成本合成(100)个32碱基的寡聚糖。这些寡核苷酸是全长的DNA，经过退火和连接，形成了编码活性tet基因的1600bp的dsDNA片段。在第一阶段，将进行实验，以验证大阵列寡聚合成方法使用800反应井原型。第一阶段：目标1是开发一种软件算法，生成图形网点图案文件，以生产用于光刻阀门工艺的丝网或激光蚀刻垫。目标2是生成和测试更大规模的光刻垫，并确定试剂的应用效率和交叉污染。目标3是利用这项技术同时合成800个寡聚糖。产生的重叠寡核苷酸将被退火和连接，形成一个单一的基因片段，该基因片段可以克隆到质粒载体中，随后进行测序，以验证整个过程。目标4将确定大阵列低聚物的试剂成本，并评估该技术作为产品或服务的总体可行性。在第二阶段，该方法将扩大到每次合成多达10,000个寡聚糖。据预测，这项技术将产生大量寡核苷酸，从而产生dsDNA，每个碱基对的成本为0.01美元，比目前供应商的价格至少低十分之一。这项工作产生的产品涉及快速增长的基因组、代谢途径和蛋白质结构操纵领域，这些领域对药物发现和开发非常重要。据估计，该产品的市场年销售额为4,000万至5,000万美元。由于麦康奈尔研究公司已经制造和销售用于分子生物学研究的仪器和试剂盒，我们公司可以在第二阶段之后直接销售这项工作开发的产品和服务。 与公共卫生相关：将进一步开发和测试一种新型的大阵列寡核苷酸合成器，该合成器允许在12小时内同时合成数万个寡核苷酸，成本不到当前技术的四分之一。这项工作开启了一个重要的合成生物学新时代的大门，在这个时代，可以完成大DNA片段的设计、合成和移植，从而生产许多新的蛋白质、药物、有用的细菌和其他研究工具。

DESCRIPTION (provided by applicant): Phase I is the development of a large-array oligonucleotide synthesis instrument that will greatly decrease the cost and simplify construction of mega base-size double-stranded DNA segments. This novel technology will use less than 1/40th of the synthesis reagents, as compared to conventional DNA synthesizers, and will produce oligos in the 100 to 500 nanogram range per reaction. The process enables the synthesis of up to 10,000 overlapping oligos that can be assembled into double-stranded DNA by annealing and ligation. The technology combines conventional oligo nucleotide synthesis chemistry with novel lithographic valving technology to deliver synthesis reagents to a large array of individual reaction chambers. In preliminary data, a prototype of this system was successfully tested. This device uses a series of computer generated valving mats that effect opening or closing of miniaturized oligo synthesis reaction chambers. The prototype allowed synthesis of (100) 32-base oligos at a reagent cost of ~$0.012 per base. These oligos were full-length, DNAs that annealed and ligated to form the predicted 1600 bp fragment of dsDNA coding for an active tet gene. In Phase I, experiments will be carried out to validate the large array oligo synthesis method using an 800-reaction well prototype. Phase I: Aim 1 is to develop a software algorithm that generates graphic dot pattern files that enable the production of silkscreen or laser etched mats to be used for the lithographic valving process. Aim 2 is to generate and test larger scale lithographic mats and determine efficiency of reagent application and cross-contamination. Aim 3 is to synthesize 800 oligos simultaneously using the technology. The resulting overlapping oligos will be annealed and ligated to form a single gene fragment that can be cloned into a plasmid vector and subsequently sequenced to validate the overall process. Aim 4 will be to determine reagent cost of a large-array oligos, and to assess the overall feasibility of the technology as a product or service. In Phase II, the methodology wil be expanded to synthesize up to 10,000 oligos per run. The technology is predicted to generate large-arrays of oligos and hence dsDNA at a cost of <$0.01 per base pair, which is at least ten fold less than current suppliers' prices. The products that result from this work address the rapidly growing field of genome, metabolic pathway, and protein structure manipulation that is important to pharmaceutical drug discovery and development. The market for the product is estimated at $40-50 million annually. Since MacConnell Research already manufactures and sells instruments and kits for molecular biology research, our company can directly market products and services developed by this work after Phase II. PUBLIC HEALTH RELEVANCE: A novel large-array oligonucleotide synthesizer will be further developed and tested that allows tens of thousands of oligonucleotides to be synthesized simultaneously in 12 hours time at cost of less than 1/40 of current technology. This work opens the door to a significant new era of synthetic biology in which the design, synthesis, and transplantation of large DNA segments can be accomplished allow for the production of many new proteins, drugs, useful bacteria, and other research tools.