Efficient Simultaneous Synthesis of Large Arrays of Oligonucleotides

描述（由申请人提供）：第一阶段是开发一种大阵列寡核苷酸合成仪器，该仪器将大大降低成本并简化百万碱基双链DNA片段的构建。与传统的DNA合成器相比，这种新技术将使用不到1/40的合成试剂，并且每个反应将产生100到500纳克范围内的低聚物。该过程可以合成多达10,000个重叠的寡核苷酸，这些寡核苷酸可以通过退火和连接组装成双链DNA。该技术将传统的寡核苷酸合成化学与新型光刻阀化技术相结合，将合成试剂输送到大量的单个反应室。在初步数据中，该系统的原型已成功测试。该装置使用一系列计算机生成的阀垫来影响小型低聚物合成反应室的开启或关闭。该原型允许合成（100）32个碱基的低聚物，每个碱基的试剂成本约为0.012美元。这些寡核苷酸是全长的dna，经过退火和连接形成预测的编码活性tet基因的1600bp的dsDNA片段。在第一阶段，将使用800个反应井原型进行实验以验证大阵列低聚物合成方法。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，使丝印或激光蚀刻垫的生产能够用于光刻阀化过程。目的2是生成和测试更大规模的光刻垫，并确定试剂应用和交叉污染的效率。目的3利用该技术同时合成800个寡核苷酸。所得到的重叠寡核苷酸将被退火并连接形成单个基因片段，该基因片段可以被克隆到质粒载体中，随后进行测序以验证整个过程。目标4将是确定大阵列寡核苷酸的试剂成本，并评估该技术作为产品或服务的整体可行性。在第二阶段，该方法将扩展到每次运行合成多达10,000个寡核苷酸。该技术预计将以每对碱基对< 0.01美元的成本产生大量寡核苷酸和双链dna，这比目前供应商的价格至少低10倍。从这项工作中产生的产品解决了基因组、代谢途径和蛋白质结构操纵这一快速增长的领域，这对药物的发现和开发非常重要。该产品的市场规模估计为每年4000万至5000万美元。由于MacConnell Research已经在生产和销售分子生物学研究仪器和试剂盒，我公司可以在二期后直接销售这项工作开发的产品和服务。

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.