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，促进了许多生物的基因组序列数据的可用性爆炸性增长。尽管有这些进展，但序列数据的绝大多数值尚未实现，因为常规测序的成本是过于良好的。基于毛细血管电泳的当前测序技术可能不允许成本降低刻板级。需要替代测序技术。在这里，我们建议通过实时监测DNA聚合化来使用DNA聚合酶作为快速和节俭的测序发动机。 Nanofluidics，Inc。是从康奈尔大学（Cornell University）明确建立的，以利用2个技术进步，从而实现实时单分子测序系统。第一个是一种光学限制技术，即零模式波导（ZMW），它允许在加工性DNA聚合过程中实时检测单核苷酸掺入。第二种末端磷酸荧光标记是将荧光团连接到核苷酸上的一种方法，使它们在掺入后自动从DNA链中自动去除。通过将DNA结构与荧光团禁用，该方法甚至可以使用标记的核苷酸替换100％替换，可以进行高度的加工掺入。这些技术的组合消除了对聚合酶的反应或不受阻滞的缓慢和昂贵洗涤的需求。由于聚合酶是自由运行的，因此只要聚合酶继续合成，序列读取就可以进行，这可以长达数十万个碱基。 ZMW和聚合酶都很小，并且系统没有流体或运动部件，使该技术可与高度的多路复用程度相提并论。该程序的目的是将这些技术部署在4色，实时的，多重的单分子DNA测序系统中，该系统将在2008年以50,000美元的价格实现哺乳动物基因组的测序，到2010年为1000美元。