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。化学合成 最多100个碱基对的寡核苷酸的化学合成是相对简单和确定的方法， 较长的寡核苷酸效率低且成本高，导致产物具有高累积误差和有限的 产率目前制备长DNA的方法反而产生较短的重叠DNA寡核苷酸， 将它们进行生物化学组装以产生所需的DNA序列。除了被问题搞糊涂之外 由于二级结构和重复序列的复杂性，这些方法费力、耗时且相对复杂， 贵了 该项目提出了一种DNA合成仪的发展，能够制造的DNA寡核苷酸的 超过两千个碱基对该合成器将是一个基于晶闸管的设备，大约是一个USB大小 拇指驱动器，并将采用酶催化的DNA合成。在给设备加载了几微升后 在纳米反应器单元（NRC）中，DNA将在单分子水平上合成。高保真DNA 扩增将用于在合成后产生更大量的DNA。艾瑞迪亚写了一本小说 基于工程拓扑异构酶的DNA合成的生物化学，并且已经显示了这种生物化学 以与半导体芯片中的固态纳米孔兼容。主要的创新理念是：（1） NRC中的试剂被纳米孔隔离，这样只有DNA（而不是酶）可以穿过 纳米孔，允许电泳控制顺序反应;（2）NRC酶加载过程， 其中通过适当的微流体引入装载有DNA碱基的活化酶 （3）二级DNA结构的工程化，其可以通过监测二级DNA结构来观察。 纳米孔电流，使得能够对合成反应进行真实的时间质量控制。 该项目的第一阶段将侧重于优化和表征平台的生物化学。的 第二阶段的目标是将生物化学整合到基于生物传感器的纳米孔装置中， 单分子DNA合成与实时监控。然后将使用高保真放大方法 来为使用者产生更多的DNA。Iridia公司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.