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

摘要 这项提议的目标是开发一种台式、基于喷墨的寡核苷酸合成器 使用水相、酶促DNA合成技术进行高达100万的平行合成 每天都有高质量的寡核苷酸。该系统将使密钥的性能提高10倍 与现有技术相比，衡量成本、质量和吞吐量。综合 生物化学将利用工程末端脱氧核苷酸转移酶，这些酶具有 已针对3‘氨氧基可逆终止子核苷酸的掺入进行了优化。这种方法 有几个优点，包括在合成寡核苷酸方面被证明具有高达 微珠上有300个核苷酸。在这里，我们将调整这项技术，使DNA合成能够在 使用喷墨喷墨进行精确试剂输送的功能化显微镜载玻片。一个多学科的 将遵循方法，在仪器工程方面向目标迈进 开发、生物化学和方案优化，以及性能基准 人工合成的寡核苷酸。在第一年，我们将构建一个初始试验台，以实现 对单个组件、材料、试剂和方案进行验证和优化。在 第二年，我们将建立一个完全自动化的原型仪器，并完善协议和 允许合成长寡核苷酸的软件。最后，在第3年，我们将构建多个 台式仪器，并与合作实验室合作，在几个 “真实世界”应用程序。随后这些工具的商业化将会民主化。 获得快速高密度寡核苷酸合成，并革命性地实现广泛的 各种合成生物学和功能基因组学应用。

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.