Sequencing by Direct Electrical Measurements of Polymerase Fluctuations

项目摘要 我们的目标是建立一个低成本，高速度的单分子基因组测序仪，具有长的读取，不需要 染料或标记，具有直接电子读出。该技术具有快速读取的潜力（大约 每个基因组一小时）。如果这种潜力是 要完全实现，那么在临床上使用基因组测序并提供近实时反馈可能会成为一种 现实我们的数据表明，大幅度的聚合酶波动与聚合酶活性。 我们的第一个目标是以高精度识别与每个核苷酸掺入相关的信号。这将 能够通过核苷酸的环状添加进行测序，但具有以下优点： 序列将被直接计数。我们的第二个目标是鉴定被掺入的单个核苷酸 基于在每次掺入时产生的信号的细节。这将允许测序仪在 在所有四种核苷酸磷酸存在下聚合酶的自由运行速度，使得 1万台设备可以在一小时内产生一个基因组的读数。我们的第三个目标是开发可扩展的 制造原型固态器件的技术。

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.