Sequencing by Direct Electrical Measurements of Polymerase Fluctuations

项目总结 我们的目标是创造一种低成本、高速的单分子基因组测序仪，具有长时间读取，不需要 直接电子读数的染料或标签。这项技术具有快速阅读的潜力(约为 每个基因组一小时)使用基于简单的两个终端设备的集成电路芯片。如果这个潜力是 要完全实现，那么在临床上使用基因组测序并提供接近实时的反馈可能成为一种 现实。我们的数据显示，大幅度的聚合酶波动与聚合酶活性有关。 我们的第一个目标是高精度地识别与每个核苷酸掺入相关的信号。这将会 能够通过核苷酸的循环加成进行测序，但具有均聚物运行的优势 序列将被直接计数。我们的第二个目标是确定被结合的单个核苷酸 基于在每次合并时产生的信号的细节。这将允许定序器在 聚合酶在所有四种核苷酸有害磷酸盐存在时的自由运行速度，因此晶片 一万台设备可以在一小时内产生相当于一个基因组的读数。我们的第三个目标是开发可伸缩的 用于制造固态器件原型的技术。

PROJECT SUMMARY Our goal is to create a low-cost, high speed single-molecule genome sequencer with long reads, requiring no dyes or labels, with direct electronic readout. The technology has the potential for rapid reads (on the order of an hour per genome) using an integrated circuit chip based on simple two terminal devices. If this potential were to be fully realized, then use of genome sequencing in the clinic with near real-time feedback could become a reality. Our data shows that large-amplitude polymerase fluctuations are associated with polymerase activity. Our first goal is to identify signals associated with each nucleotide incorporation with high accuracy. This would enable sequencing by means of cyclic addition of nucleotides, but with the advantage that homopolymer runs of sequence would be counted directly. Our second aim is to identify the individual nucleotides being incorporated based on the details of the signals generated at each incorporation. This would allow a sequencer to run at the free-running speed of the polymerase in the presence of all four nucleotidetriphosphates, so that a wafer of 10,000 devices could produce a genome's worth of reads in an hour. Our third aim is to develop scalable technology for fabrication of prototype solid-state devices.