High throughput low cost DNA sequencing using probe tip arrays

使用探针阵列进行高通量低成本 DNA 测序

基本信息

批准号：
7668065
负责人：
Hemantha Kumar Wickramasinghe
金额：
$ 70万
依托单位：
UNIVERSITY OF CALIFORNIA-IRVINE
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-08-01 至 2010-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7668065
关键词：
Address Bacteriophage lambda Base Sequence Benchmarking Biotechnology Blood capillaries California Capillary Electrophoresis Collaborations DNA DNA Sequence Detection Development Electrical Engineering Electrophoresis Fluorescence Genome Goals Hand Human Genome Label Light Measurement Medicine Methodology Methods Microscope Molecular Nanotechnology Nature Operative Surgical Procedures Optics Principal Investigator Reaction Reading Reagent Research Research Personnel Resolution Sample Size Sampling Scanning Scanning Probe Microscopes Scheme Sorting - Cell Movement Source Speed Surface Techniques Technology Therapeutic Time Universities Work base capillary cost experience fluorophore high throughput analysis instrumentation mammalian genome meetings multidisciplinary nanofabrication nanoscale novel novel strategies optical imaging programs research study scale up skills success two-dimensional

项目摘要

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.

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。限制进展的一个主要因素是当前技术的固有成本达到每个哺乳动物基因组数百万美元。我们的方法基于经过验证的 DNA 测序桑格方法的新颖实现。我们依靠纳米技术来大幅提高通量、减少试剂用量并将成本降低几个数量级。在前期工作中，我们已经证明了沿着原子力显微镜探针尖端电泳分离 DNA 片段的新方法的可行性。我们表明，基于这种表面电泳方法，可以将多达至少 100 个碱基的链分离，样品数量减少到只有几个分子，并且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。桑格反应产生的荧光标记 DNA 片段按大小分类成条带。我们使用一种新颖的光学检测方案检测它们在探针末端的通过。我们设想在 100x100 探针阵列中实现大规模并行操作模式。我们的目标是开发一种技术，通过具有高分析通量和最终极少量试剂的探针尖端测序仪的并行操作，最终达到每个基因组 1000 美元的目标。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电泳分离高达 100bp 的 DNA 片段的 DNA 测序方案。目标 2. 为了证明可扩展性，我们使用 10 个探针尖端的线性阵列解决 DNA 测序仪的并行操作问题。