Real-time Multiplex Single-Molecule DNA Sequencing

描述(申请人提供)：基因组测序给生物学和医学带来了革命性的变化。在过去的10年里，测序成本下降了5倍，推动了无数生物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高得令人望而却步。目前基于毛细管电泳法的测序技术很可能无法实现成本的数量级下降。需要替代的测序技术。在这里，我们建议使用DNA聚合酶作为一个快速和廉价的测序引擎，通过实时监测DNA聚合。纳米流体公司是从康奈尔大学剥离出来成立的，明确地利用了两项技术进步，使实时单分子测序系统成为可能。第一种是光学限制技术，即零模波导(ZMW)，它允许在DNA聚合过程中实时检测单核苷酸掺入。第二种是末端磷酸盐荧光标记，是一种将荧光团连接到核苷酸上的方法，使它们在掺入后自动从DNA链上移除。通过使DNA结构不受荧光团的阻碍，即使用标记的核苷酸100%替换，这种方法也允许高度进行掺入。这些技术的结合消除了缓慢且昂贵的反应洗涤或解锁聚合酶的需要。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，合成的碱基可能长达数十万个碱基。ZMW和聚合酶都很小，而且系统没有流体或运动部件，使该技术适合于高度多路复用。该计划的目标是将这些技术部署在四色、实时、多路复用单分子DNA测序系统中，到2008年将能够以5万美元的价格对哺乳动物基因组进行测序，到2010年将以1000美元的价格进行测序。

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.