Development of an Accurate, Long and Fast DNA Synthesis Device

摘要 近年来，对长定制合成DNA的需求迅速增加。而化学合成 最多100个碱基对的寡核苷酸是一种相对简单和成熟的过程，化学合成 较长的寡核苷酸效率低且成本高，导致产品具有高累积误差和有限的 投降。目前制造长DNA的方法转而产生较短的重叠DNA寡核苷酸和 用生物化学的方法将它们组装起来，产生所需的DNA序列。除了被问题搞糊涂之外 对于二级结构和重复序列，这些方法费力、耗时且相对 很贵的。 本项目提出了一种能够制造DNA寡核苷酸的DNA合成器的开发 超过2000个碱基对。合成器将是一个基于半导体的设备，大约有USB大小 拇指驱动器，并将采用酶催化的DNA合成。在给设备装入几微升后 在试剂中，DNA将在单分子水平上在纳米反应器单元(NRC)中合成。高保真的DNA 扩增将被用来在合成后产生更多的DNA。伊里迪亚开发了一部小说 基于工程拓扑异构酶的DNA合成的生物化学，并展示了这种生物化学 与半导体芯片中的固态纳米孔兼容。关键的创新概念是：(1) NRC中的试剂由纳米孔隔离，因此只有DNA(而不是酶)可以通过 纳米孔，允许对顺序反应进行电泳性控制；(2)NRC酶负载过程， 其中带有DNA碱基的激活酶通过适当的微流控被引入 通道；和(3)二级DNA结构的工程，可以通过监测 纳米孔电流，实现合成反应的实时质量控制。 该项目的第一阶段将侧重于优化和表征平台的生物化学。这个 第二阶段的目标是将生物化学集成到基于半导体的纳米孔设备中，以实现 具有实时监控的单分子DNA合成。然后将使用高保真放大方法 为用户产生更大数量的DNA。Iridia，Inc.希望这种方法能够合成DNA 几千个核苷酸，错误率非常低。

ABSTRACT In recent years the need for long custom synthetic DNA has rapidly increased. While chemical synthesis of oligonucleotides of up to 100 base pairs is a relatively simple and established process, chemical synthesis of longer oligonucleotides is inefficient and costly, resulting in a product with high accumulated errors and limited yield. Current approaches to making long DNAs instead generate shorter overlapping DNA oligos and biochemically assemble them to create the desired DNA sequence. In addition to being confounded by issues of secondary structure and repetitive sequences, these methods are laborious, time-consuming and relatively expensive. This project proposes the development of a DNA synthesizer capable of manufacturing DNA oligonucleotides of over two-thousand base pairs. The synthesizer will be a semiconductor-based device, about the size of a USB thumb-drive, and will employ enzyme-catalyzed DNA synthesis. After loading the device with a few microliters of reagent, DNA will be synthesized at the single molecule level in a nano reactor cell (NRC). A high-fidelity DNA amplification will be used to generate larger quantities of DNA post synthesis. Iridia has developed a novel biochemistry for the DNA synthesis based on an engineered topoisomerase, and have shown this biochemistry to be compatible with solid-state nanopores in a semiconductor chip. The key innovation concepts are: (1) the reagents in the NRC are segregated by nanopores, such that only DNA (and not enzymes) can move through the nanopores, allowing electrophoretic control of the sequential reactions; (2) the NRC enzyme loading process, wherein activated enzymes charged with DNA bases are introduced through the appropriate microfluidic channels; and (3) engineering of a secondary DNA structure which can be observed via monitoring of the nanopore current, enabling real time quality control of the synthesis reaction. The first phase of this project will focus on optimizing and characterizing the biochemistry of the platform. The second phase will be aimed to integrate the biochemistry into a semiconductor-based nanopore device to enable single molecule DNA synthesis with real-time monitoring. A high-fidelity amplification approach will then be used to generate larger quantities of DNA for the user. Iridia, Inc. expect this approach to enable synthesis of DNA of several thousand nucleotides with a very low error rate.