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 理解基因组生物学和遗传变异对个体健康的后果的能力 这在很大程度上取决于让研究人员能够读取和写入DNA的技术。测序技术 最近已经看到了巨大的进步，使个人基因组负担得起的几乎任何 实验室，甚至直接提供给消费者。然而，相应的DNA合成技术却滞后于 远远落后于这些发展，导致了合成生物学研究基因的主要障碍， 变体和感兴趣的基因组。这项提议旨在开发一种新的DNA合成方法 技术，以解决当前平台面临的最大挑战：保持足够的准确性， 高精度应用和大规模应用的吞吐量，同时保持成本效益， 可访问性。为了实现这一点，传统的合成DNA寡核苷酸的亚磷酰胺方法 将被适配到纳米颗粒上，纳米颗粒将通过含有合成物的液滴移动， 沿着等离子体表面阵列。这条“传送带”将通过C形光学控制， 雕刻（CSE）集中来自下方的光，用作光学陷阱。这样，珠子和 试剂液滴可以单独地、快速地输送到多个通道中的特定光阱， 改变照明波长，使数百万个独特的寡核苷酸得以合成， 同时在一个数组中。通过根据以下因素定制试剂液滴尺寸和浓度， 合成规模，该方法将被优化，以靶向整个珠表面，最大限度地提高产量，并消除 试剂使用过量。将通过测试不同DNA序列的合成来评估质量。主要 这种新型DNA合成平台的优点包括：1）更快的反应（循环时间45秒），2）更低的 由于酸暴露降低的错误率（<1：1000），3）高产率（>5阿托摩尔/珠），4）增加的长度 由于更清洁的合成，寡核苷酸（>300个碱基），6）显著减少危险废物的产生，7） 在单次运行中产生> 2500万个独特的寡核苷酸序列，其可以单独分离用于 下游应用，以及8）0.0000001美元/基的成本，比最小值小两个数量级 昂贵的方法目前可用。具有这些特性的DNA合成技术将使 前所未有的基因组研究，使研究人员能够测试功能和临床影响， 数以千计的基因和遗传变异。