Real-time Multiplex Single-Molecule DNA Sequencing

实时多重单分子 DNA 测序

• 批准号: 7103689
• 负责人: STEPHEN WHITFIELD TURNER
• 金额: $ 216.25万
• 依托单位: PACIFIC BIOSCIENCES OF CALIFORNIA, INC.
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-08-01 至 2008-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7103689
• 关键词: DNA directed DNA polymerase; biomedical automation; biotechnology; clinical research; fluorescent dye /probe; human genetic material tag; nanotechnology; nucleic acid sequence; optics; technology /technique development

DESCRIPTION (provided by applicant): Genome sequencing has revolutionized biology and medicine. A 5-fold decrease in sequencing cost over the past 10 years has fueled an explosive growth in the availability of genome sequence data for numerous organisms. Despite these advances, the vast majority of the value from sequence data has yet to be realized, as the cost of routine sequencing is prohibitive. Current sequencing technologies based on capillary electrophoresis will likely not allow order-of-magnitude decreases in cost. Alternative sequencing technologies are required. Here we propose to use DNA polymerase enzyme as a fast and frugal sequencing engine by monitoring DNA polymerization in real-time. Nanofluidics, Inc. was established as a spin-out from Cornell University explicitly to leverage 2 technological advances that enable real-time single-molecule sequencing system. The first is an optical confinement technology, the zero-mode waveguide (ZMW), which allows detection of single nucleotide incorporation in real-time during processive DNA polymerization. The second, terminal-phosphate fluorescent labeling, is a method of attaching fluorophores to nucleotides such that they are automatically removed from the DNA strand after incorporation. By leaving the DNA structure un-hindered with fluorophores, this method allows highly processive incorporation even using 100% replacement with labeled nucleotides. The combination of these technologies eliminates the need for slow and expensive washing of the reaction or un-blocking of the polymerase. Because the polymerase is free-running, the sequence read can proceed as long as the polymerase continues synthesizing, which can be as long as hundreds of thousands of bases. Both the ZMW and the polymerase are small, and the system has no fluidics or moving parts, making the technology amenable to high degrees of multiplexing. The goal of this program is to deploy these technologies in a 4-color, real-time, multiplex single-molecule DNA sequencing system that will enable sequencing of a mammalian genome for $50,000 by 2008, and $1000 by 2010.

描述（由申请人提供）：基因组测序已经彻底改变了生物学和医学。在过去的10年中，测序成本下降了5倍，这推动了许多生物体基因组序列数据的爆炸性增长。尽管取得了这些进展，但序列数据的绝大多数价值尚未实现，因为常规测序的成本过高。目前基于毛细管电泳的测序技术可能不允许成本的数量级下降。需要替代测序技术。在这里，我们建议使用DNA聚合酶作为一个快速和节俭的测序引擎，通过实时监测DNA聚合。Nanofluidics公司是作为康奈尔大学的一个分支而建立的，明确地利用了2项技术进步，使实时单分子测序系统成为可能。第一种是光学限制技术，零模波导（ZMW），它允许在进行性DNA聚合过程中实时检测单核苷酸掺入。第二，末端磷酸荧光标记，是一种将荧光团连接到核苷酸上的方法，这样它们在掺入后自动从DNA链上除去。通过使DNA结构不受荧光团的阻碍，该方法允许高度进行性掺入，即使使用标记核苷酸的100%替换。这些技术的组合消除了对反应的缓慢且昂贵的洗涤或聚合酶的解封闭的需要。因为聚合酶是自由运行的，所以只要聚合酶继续合成，序列读取就可以进行，其可以长达数十万个碱基。ZMW和聚合酶都很小，并且该系统没有流体或移动部件，使得该技术适用于高度的多路复用。该计划的目标是在四色、实时、多重单分子DNA测序系统中部署这些技术，到2008年，该系统将使哺乳动物基因组测序的成本达到5万美元，到2010年达到1000美元。