A low-cost benchtop oligonucleotide synthesizer using inkjet enzymatic DNA synthesis

使用喷墨酶法 DNA 合成的低成本台式寡核苷酸合成仪

• 批准号: 10213239
• 负责人: ADRIAN HORGAN
• 金额: $ 73.45万
• 依托单位: DNA SCRIPT
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10213239
• 关键词: Area; Benchmarking; Biochemistry; Biology; Biomedical Research; Biotechnology; Biotin; Businesses; Chemicals; Chemistry; Complementary DNA; Computer software; Cost Measures; DNA; DNA Nucleotidylexotransferase; DNA biosynthesis; Development; Engineering; Enzymes; Formulation; Goals; Hybrids; Individual; Laboratories; Length; Microscope; Microspheres; Modeling; Mutagenesis; Nucleotides; Oligonucleotides; Performance; Price; Protocols documentation; Reagent; Reproducibility; Running; Services; Slide; Synthesis Chemistry; System; Technology; Testing; Time; Work; aqueous; base; chemical synthesis; commercialization; cost; density; functional genomics; instrument; instrumentation; interdisciplinary approach; novel; operation; prototype; real world application; synthetic biology; synthetic genomics; transcriptomics; user-friendly

ABSTRACT The objective of this proposal is to develop a benchtop, inkjet-based, oligonucleotide synthesizer using aqueous, enzymatic DNA synthesis technologies for the parallel synthesis of up to 1 million high-quality oligonucleotides per day. The system will deliver >10-fold improvements in the key metrics of cost, quality, and throughput compared to existing technologies. The synthesis biochemistry will utilize engineered terminal deoxynucleotidyl transferase enzymes that have been optimized for incorporation of 3’ aminoxy reversible terminator nucleotides. This approach has several advantages, including proven performance in the synthesis of oligonucleotides up to 300 nucleotides on microbeads. Here, we will adapt the technology to enable DNA synthesis on functionalized microscope slides using inkjets for precise reagent delivery. A multidisciplinary approach will be followed to advance toward the goals in terms of engineering for instrumentation development, biochemistry and protocol optimization, and performance benchmarking of the synthesized oligonucleotides. During the first year, we will build an initial testbed to enable verification and optimization of individual components, materials, reagents, and protocols. In the second year, we will build a fully automated prototype instrument, and refine the protocols and software to allow synthesis of long oligonucleotides. Finally, Year-3, we will build multiple benchtop instruments and work with partner laboratories to test the oligonucleotides in several ‘real world’ applications. Subsequent commercialization of the instruments will democratize access to rapid high-density oligonucleotide synthesis, and revolutionize approaches to a wide variety of synthetic biology and functional genomics applications.

摘要 本提案的目的是开发一种台式、基于喷墨的寡核苷酸合成仪 使用水相酶促DNA合成技术，平行合成多达100万个 高质量的寡核苷酸。该系统将提供>10倍的改进， 与现有技术相比，成本、质量和吞吐量指标。合成 生物化学将利用工程末端脱氧核苷酸转移酶， 已被优化用于掺入3'氨氧基可逆终止子核苷酸。这种方法 具有几个优点，包括在寡核苷酸合成中的经证实的性能， 微珠上有300个核苷酸在这里，我们将调整技术，使DNA合成上 功能化显微镜载玻片使用喷墨精确试剂输送。一个多学科 在仪器仪表工程方面，将遵循一种方法，朝着目标前进 开发、生物化学和方案优化，以及 合成的寡核苷酸。在第一年，我们将建立一个初始测试平台， 单个组件、材料、试剂和方案的验证和优化。在 第二年，我们将建立一个完全自动化的原型仪器，并完善协议， 软件以允许合成长寡核苷酸。最后，第三年，我们将建立多个 台式仪器，并与合作伙伴实验室合作，在几个国家测试寡核苷酸， “真实的世界”应用程序。这些工具随后的商业化将使 获得快速高密度寡核苷酸合成，并彻底改变方法， 多种合成生物学和功能基因组学应用。