Ultra high-throughput DNA synthesis via nano-optical conveyer belts

通过纳米光学传送带进行超高通量 DNA 合成

• 批准号: 9379771
• 负责人: Ronald Wayne Davis
• 金额: $ 30.19万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-09 至 2019-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9379771
• 关键词: Acids; Address; Agriculture; Automation; Biology; Caliber; Cleaved cell; Clinical; DNA; DNA Sequence; DNA biosynthesis; DNA sequencing; Development; Diploidy; Disease; Electrical Engineering; Engravings; Ethics; Generations; Genes; Genetic Variation; Genome; Genomic medicine; Genomics; Guanine + Cytosine Composition; Hazardous Waste; Health; Human Genome; Human Genome Project; Individual; Industry Standard; Investigation; Laboratories; Lasers; Length; Light; Lighting; Location; Methods; Nucleotides; Oligonucleotides; Optics; Physiologic pulse; Polystyrenes; Production; Property; Reaction; Reagent; Recording of previous events; Research Personnel; Running; Sampling; Speed; Surface; Technology; Temperature; Testing; Time; To specify; Writing; base; cost; cost effective; cost effectiveness; gene function; genetic variant; genome editing; interest; melting; nano; nanoparticle; novel; optical traps; phosphoramidite; plasmonics; precision medicine; qubit; social implication; synthetic biology; synthetic construct; tool; virtual

PROJECT SUMMARY The ability to understand genome biology and the consequences of genetic variation on individual health depends heavily on technologies that allow researchers to read and write DNA. Sequencing technologies have recently seen dramatic improvements that have made personal genomes affordable for virtually any laboratory and even directly available to consumers. Yet the corresponding DNA synthesis technologies lag far behind these developments, causing a major hindrance in synthetic biology efforts to study genes, variants, and genomes of interest by synthesizing them. This proposal aims to develop a novel DNA synthesis technology to address the greatest challenge faced by current platforms: maintaining sufficient accuracy for precision applications and throughput for large-scale applications while remaining cost-effective for accessibility. To achieve this, the traditional phosphoramidite method of synthesizing DNA oligonucleotides will be adapted onto nanoparticular beads, which will be moved through droplets containing synthesis reagents along a plasmonic surface array. This `conveyer belt' will be optically controlled via C-shaped engravings (CSEs) that concentrate light from below, serving as optical traps. In this way, the beads and reagent droplets can be individually, rapidly transported to specific optical traps in multiple lanes simply by changing the illumination wavelengths, allowing millions of unique oligonucleotides to be synthesized simultaneously on a single array. By tailoring the reagent droplet size and concentration depending on synthesis scale, the method will be optimized to target the entire bead surface, maximize yield, and eliminate excess reagent usage. Quality will be assessed by testing synthesis of diverse DNA sequences. The main advantages of this novel DNA synthesis platform will include: 1) faster reactions (cycle time 45 sec), 2) lower error rate due to decreased acid exposure (<1:1000), 3) high yield (>5 attomoles/bead), 4) increased length of oligonucleotides (>300 bases) due to cleaner synthesis, 6) significantly less hazardous waste production, 7) generation of >25 million unique oligonucleotide sequences in a single run that can be individually isolated for downstream applications, and 8) a cost of $0.0000001/base, two orders of magnitude less than the least expensive method currently available. A DNA synthesis technology with these properties will enable unprecedented genomic investigations, allowing researchers to test the functional and clinical impact of thousands of genes and genetic variants.

项目摘要 了解基因组生物学的能力以及遗传变异对个体健康的后果 在很大程度上取决于允许研究人员读写DNA的技术。测序技术 最近已经看到了戏剧性的改进，使个人基因组几乎负担得起 实验室，甚至直接可供消费者使用。然而，相应的DNA合成技术滞后 远远落后于这些事态发展，在合成生物学研究基因的努力中造成了重大障碍， 通过合成它们的变体和感兴趣的基因组。该建议旨在开发一种新型的DNA合成 解决当前平台面临的最大挑战的技术：保持足够的准确性 大规模应用的精确应用和吞吐量，同时保持成本效益 可访问性。为此，传统的磷光岩合成DNA寡核苷酸的方法 将被调整到纳米珠珠中，该珠将通过包含合成的液滴移动 沿等离子体表面阵列的试剂。此“传送带”将通过C形光学控制 从下方集中光的版画（CSE），用作光学陷阱。这样，珠子和 试剂液滴可以单独，快速运输到多个车道中的特定光学陷阱 更改照明波长，可以合成数百万个独特的寡核苷酸 同时在一个数组上。通过调整试剂液滴的尺寸和浓度，具体取决于 合成量表，该方法将被优化以靶向整个珠表面，最大化产量并消除 过量的试剂用法。质量将通过测试各种DNA序列的合成来评估。主 这个新型DNA合成平台的优点将包括：1）更快的反应（周期时间45秒），2）较低 由于酸暴露降低引起的错误率（<1：1000），3）高产量（> 5 attomoles/珠），4）增加长度的增加 由于清洁剂的合成，寡核苷酸（> 300个碱基），6）危险废物产生少得多，7） 在单个运行中产生> 2500万个独特的寡核苷酸序列，可以单独隔离为 下游应用程序和8）成本为$ 0.0000001/base，两个数量级比最小少。 当前可用的昂贵方法。具有这些特性的DNA合成技术将启用 前所未有的基因组研究，使研究人员能够测试 成千上万的基因和遗传变异。