Efficient Simultaneous Synthesis of Large Arrays of Oligonucleotides

高效同时合成大量寡核苷酸

• 批准号: 8310917
• 负责人: WILLIAM P MACCONNELL
• 金额: $ 30万
• 依托单位: MACCONNELL RESEARCH CORPORATION
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-05-22 至 2013-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8310917
• 关键词: Address; Algorithms; Amino Acid Sequence; Bacteria; Base Pairing; Code; Computer software; Consumption; DNA; DNA Sequence; DNA annealing; DNA biosynthesis; Data; Development; Devices; Genes; Genome; Hour; Individual; Lasers; Length; Ligation; Marketing; Measurement; Metabolic Pathway; Methodology; Methods; Modification; Molecular Biology; Oligonucleotide Microarrays; Oligonucleotides; Pattern; Peptide Sequence Determination; Pharmaceutical Preparations; Pharmacologic Substance; Phase; Plasmid Cloning Vector; Price; Printing; Procedures; Process; Production; Proteins; Reaction; Reagent; Research; Running; Series; Services; Synthesis Chemistry; System; Technology; Testing; Tetanus Helper Peptide; Tetracycline Resistance; Time; Transplantation; Work; base; computer generated; cost; design; drug development; drug discovery; ds-DNA; instrument; miniaturize; new technology; novel; nucleotide metabolism; prevent; protein structure; prototype; research study; synthetic biology; tool

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.

描述（申请人提供）：第一阶段是大阵列寡核苷酸合成仪器的开发，该仪器将大大降低成本并简化兆碱基大小双链DNA片段的构建。与传统的DNA合成仪相比，这种新技术将使用不到1/40的合成试剂，并且每次反应将产生100至500纳克范围内的寡核苷酸。该过程能够合成多达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技术相结合，将合成试剂输送到大量单独的反应室。在初步数据中，该系统的原型已成功测试。该装置使用一系列计算机生成的阀垫，其实现小型化寡聚合成反应室的打开或关闭。该原型允许以每个碱基约0.012美元的试剂成本合成（100）个32碱基的寡核苷酸。这些寡核苷酸是全长DNA，其退火并连接以形成预测的编码活性泰特基因的dsDNA的1600 bp片段。 在第一阶段，将进行实验，以验证使用800反应孔原型的大阵列寡核苷酸合成方法。第一阶段：目标1是开发一种软件算法，生成图形点图案文件，使生产丝网或激光蚀刻垫用于平版阀工艺。目的2是产生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。将所得重叠寡核苷酸退火并连接以形成单个基因片段，其可以克隆到质粒载体中并随后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，该方法将扩大到每次运行合成多达10，000个寡核苷酸。预计该技术将以每碱基对<0.01美元的成本产生大阵列的寡核苷酸，从而产生双链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快速增长的基因组，代谢途径和蛋白质结构操纵领域，这对药物发现和开发至关重要。该产品的市场估计每年为4000万至5000万美元。由于MacConnell Research已经生产和销售用于分子生物学研究的仪器和试剂盒，因此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。 公共卫生关系：一种新型的大阵列寡核苷酸合成仪将被进一步开发和测试，它允许在12小时内同时合成数万种寡核苷酸，成本不到现有技术的1/40。这项工作为合成生物学的一个重要新时代打开了大门，在这个新时代中，可以完成大DNA片段的设计、合成和移植，从而生产出许多新的蛋白质、药物、有用的细菌和其他研究工具。