Real-time Multiplex Single-Molecule DNA Sequencing

实时多重单分子 DNA 测序

• 批准号: 7921327
• 负责人: STEPHEN WHITFIELD TURNER
• 金额: $ 71.44万
• 依托单位: PACIFIC BIOSCIENCES OF CALIFORNIA, INC.
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7921327
• 关键词: Adhesions; Adopted; Algorithms; Bacterial Genome; Base Sequence; Biology; Capillary Electrophoresis; Collaborations; Color; DNA; DNA Sequence; DNA Structure; DNA-Directed DNA Polymerase; Data; Detection; Development; Devices; Directed Molecular Evolution; Electronics; Enzymes; Evaluation; Genome; Goals; Growth; Immobilization; Kinetics; Label; Lasers; Left; Length; Licensing; Lighting; Medicine; Methods; Monitor; Nucleotides; Optics; Organism; Performance; Phase; Point Mutation; Polymerase; Property; Reaction; Reading; Running; Sampling; Signal Transduction; Source; Speed; Surface; System; Techniques; Technology; Time; Universities; Validation; base; cost; data acquisition; detector; digital; fluorophore; genome sequencing; improved; inorganic phosphate; mammalian genome; nanofluidic; nucleotide analog; polymerization; programs; single molecule

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。过去 10 年来，测序成本降低了 5 倍，推动了众多生物体基因组序列数据的可用性爆炸性增长。尽管取得了这些进步，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令人望而却步。当前基于毛细管电泳的测序技术可能无法实现成本的数量级降低。需要替代测序技术。在这里，我们建议通过实时监测 DNA 聚合，使用 DNA 聚合酶作为快速、节俭的测序引擎。 Nanofluidics, Inc. 是康奈尔大学的衍生公司，明确利用两项技术进步来实现实时单分子测序系统。第一种是光学限制技术，即零模波导 (ZMW)，它可以在 DNA 聚合过程中实时检测单核苷酸的掺入情况。第二种是末端磷酸荧光标记，是将荧光团附着到核苷酸上的方法，以便它们在掺入后自动从 DNA 链中去除。通过使 DNA 结构不受荧光团的阻碍，该方法甚至可以使用标记核苷酸进行 100% 替换，从而实现高度持续的掺入。这些技术的结合消除了缓慢且昂贵的反应清洗或聚合酶解封闭的需要。由于聚合酶是自由运行的，只要聚合酶继续合成，序列读取就可以继续进行，合成长度可能长达数十万个碱基。 ZMW 和聚合酶都很小，并且该系统没有流体或移动部件，使得该技术适合高度多重化。该计划的目标是将这些技术部署在 4 色、实时、多重单分子 DNA 测序系统中，到 2008 年，哺乳动物基因组测序的成本将达到 50,000 美元，到 2010 年将达到 1000 美元。

项目成果

Long, processive enzymatic DNA synthesis using 100% dye-labeled terminal phosphate-linked nucleotides.

• DOI: 10.1080/15257770802260741
• 发表时间: 2008-09
• 期刊: Nucleosides, nucleotides & nucleic acids
• 作者: Korlach J;Bibillo A;Wegener J;Peluso P;Pham TT;Park I;Clark S;Otto GA;Turner SW
• 通讯作者: Turner SW

A flexible and efficient template format for circular consensus sequencing and SNP detection.

• DOI: 10.1093/nar/gkq543
• 发表时间: 2010-08
• 期刊: Nucleic acids research
• 影响因子: 14.9
• 作者: Travers KJ;Chin CS;Rank DR;Eid JS;Turner SW
• 通讯作者: Turner SW