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

项目总结 理解基因组生物学和基因变异对个体健康影响的能力 这在很大程度上依赖于允许研究人员读写DNA的技术。测序技术 最近看到了戏剧性的进步，使得几乎所有人都能负担得起个人基因组 实验室甚至直接提供给消费者。然而，相应的DNA合成技术却滞后了 远远落后于这些发展，造成了合成生物学研究基因的主要障碍， 变异，以及通过合成它们而感兴趣的基因组。这项提议旨在开发一种新的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 精确的应用程序和大规模应用程序的吞吐量，同时保持经济高效的 可访问性。为了实现这一点，传统的亚磷酰胺合成DNA寡核苷酸的方法 将被改编到纳米颗粒珠子上，这些珠子将通过含有合成的液滴 沿着等离子体表面阵列的试剂。这条“传送带”将通过C型进行光学控制 作为光学陷阱的从下面聚集光线的雕刻(CSE)。通过这种方式，珠子和 只需通过以下操作即可将试剂液滴单独、快速地传输到多个通道中的特定光学陷阱 改变光照波长，可以合成数百万种独特的寡核苷酸 同时在单个阵列上。通过根据需要调整试剂液滴的大小和浓度 合成规模，该方法将被优化为针对整个珠粒表面，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 这种新型的DNA合成平台的优点包括：1)反应速度更快(周期为45秒)，2)更低 由于酸暴露减少而导致的错误率(&lt；1：1000)，3)高产量(&gt；5个attomoles/珠子)，4)增加长度 由于更清洁的合成，寡核苷酸(&gt；300个碱基)，6)显著减少的危险废物产生，7) 在一次运行中生成2500万个唯一的寡核苷酸序列，这些序列可以单独分离用于 下游应用，8)成本为0.0000001美元/基础，比最低成本低两个数量级 目前可用的方法成本很高。具有这些特性的DNA合成技术将使 史无前例的基因组研究，使研究人员能够测试其功能和临床影响 数以千计的基因和基因变异。

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.