High throughput low cost DNA sequencing using probe tip arrays

描述(申请人提供)：人类基因组测序在医学和生物技术领域产生了巨大影响，并开辟了个人治疗领域。一个主要的进展限制因素是当前技术的固有成本，每个哺乳动物基因组的成本高达数百万美元。我们的方法是基于一种新的实施证明的DNA测序桑格方法。我们依靠纳米技术来极大地提高产量，减少试剂用量，并将成本降低数量级。在初步的工作中，我们已经证明了一种新的方法的可行性，即沿着原子力显微镜探针尖端的表面进行DNA片段的电泳法分离。我们发现，基于这种表面电泳法，可以分离出至少100个碱基的链，样本量只有几个分子，而且速度比传统的毛细管电泳快四到五个数量级。我们现在建议将这一方法发展为基于桑格方法的完整DNA测序方案。桑格反应产生的荧光标记的DNA片段按大小分类为条带。我们使用一种新的光学检测方案来检测它们在探头末端的通过。我们设想了一种在100x100探头尖端阵列中的大规模并行操作模式。我们的目标是开发一种技术，通过并行操作具有高分析吞吐量和最终非常少量的试剂的探针尖端测序仪，最终达到每个基因组1000美元的目标。本研究的具体目的如下：目的1.提出了一种基于单探针尖电泳法的DNA测序方案。目的2.为了展示可伸缩性，我们使用10个探针头的线性阵列来解决DNA测序仪的并行操作问题。

DESCRIPTION (provided by applicant): Sequencing of the human genome created a large impact in medicine and biotechnology and opened up the realm of personal therapeutics. A major progress-limiting factor is the inherent cost of current technologies reaching millions of dollars per mammalian genome. Our approach is based on a novel implementation of the proven Sanger method for DNA sequencing. We rely on nanotechnology to greatly increase throughput, reduce reagent volumes and drive costs down by orders of magnitude. In preliminary work, we have demonstrated the feasibility of a new approach for electrophoretic separation of DNA fragments along the surface of an Atomic Force Microscope probe tip. We showed that based on this surface electrophoresis method strands up to at least 100 bases can be separated in size with sample quantities down to only a few molecules and with speeds that are four to five orders of magnitude faster than in conventional capillary electrophoresis . We now propose to evolve this methodology toward a complete DNA sequencing scheme based on the Sanger method. Fluorescently labeled DNA fragments resulting from the Sanger reactions are sorted by size into bands . We detect their passage at the end of the probe tip using a novel optical detection scheme. We envision a massively parallel mode of operation in an array of 100x100 probe tips. We aim to develop technology that can ultimately reach the goal of $1000 per genome by the parallel operation of probe tip sequencers with high analysis throughput and ultimately very small volumes of reagents. The specific aims of this research are the following: Aim 1. We demonstrate a scheme for DNA sequencing based on single probe tip electrophoretic separation of DNA fragments up to 100bp. Aim 2. To demonstrate scalability, we address the parallel operation of DNA sequencers with a linear array of 10 probe tips.