High throughput low cost DNA sequencing using probe tip arrays

描述（由申请人提供）：人类基因组测序对医学和生物技术产生了巨大影响，并开辟了个人治疗领域。一个主要的进展限制因素是目前技术的固有成本达到每个哺乳动物基因组数百万美元。我们的方法是基于一个新的实施证明桑格方法的DNA测序。我们依靠纳米技术来大大提高通量，减少试剂体积，并将成本降低几个数量级。在初步工作中，我们已经证明了一种新的方法的可行性，用于电泳分离DNA片段沿着表面的原子力显微镜探针尖端。我们表明，基于这种表面电泳方法，可以分离至少100个碱基的链，样品量仅为几个分子，速度比传统毛细管电泳快四到五个数量级。我们现在建议将这种方法发展为基于桑格方法的完整DNA测序方案。通过大小将从桑格反应中得到的双标记DNA片段分选成条带。我们使用一种新的光学检测方案检测它们在探针末端的通道。我们设想在100x100探针尖端的阵列中的大规模并行操作模式。我们的目标是开发技术，最终可以达到每个基因组1000美元的目标，通过并行操作的探针尖端测序仪具有高分析通量和最终非常小的试剂体积。本研究的具体目标如下：目标1。我们展示了一种基于单探针尖端电泳分离高达100bp的DNA片段的DNA测序方案。 目标二。为了证明可扩展性，我们解决了并行操作的DNA测序仪与线性阵列的10个探针提示。

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.