An Integrated System for Single Molecule Electronic Sequencing by Synthesis

单分子电子合成测序集成系统

• 批准号: 8728991
• 负责人: GEORGE M CHURCH
• 金额: $ 171.5万
• 依托单位: COLUMBIA UNIV NEW YORK MORNINGSIDE
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-01 至 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8728991
• 关键词: Active Sites; Area; Base Sequence; Binding; Charge; Complex; DNA; DNA Sequence; DNA Sequence Determination; DNA-Directed DNA Polymerase; Detection; Development; Diagnostic; Diffuse; Electrical Engineering; Electrodes; Electronics; Ensure; Ethylene Glycols; Feedback; Foundations; Genome; Genomics; Goals; Head; Hemolysin; Indium; Left; Length; Link; Measures; Medical; Medicine; Methodology; Methods; Modification; Mutate; Nucleic Acids; Nucleotides; Polyethylene Glycols; Polymerase; Polymers; Polynucleotides; Production; Property; Proteins; Reaction; Reading; Reporting; Scientist; Sequence Determination; Side; Signal Transduction; Speed; System; Techniques; Testing; Text; Time; Ursidae Family; Vestibule; Work; base; chemical property; constriction; cost; density; design; ethylene glycol; genome sequencing; information processing; inorganic phosphate; millisecond; mutant; nano; nanofabrication; nanopore; nucleotide analog; phosphodiester; polypeptide; prevent; prototype; research and development; research study; sensor; single molecule; tripolyphosphate; voltage

There is a great need to reduce the cost of DNA sequencing to achieve the goal of the $1000 genome. We recently developed a new nanopore-based sequencing by synthesis (Nano-SBS) approach. In this project, we will pursue the development of the Nano-SBS approach into a high throughput real-time single-molecule sequencing platform. In the Nano-SBS method, a polymer tag of distinct size and charge is attached to the terminal phosphate of each of the four nucleotides. When the complementary nucleotide analog enters a template-primer-polymerase complex that is attached to the nanopore during the polymerase reaction, the tag specific for that nucleotide is captured in the voltage gradient within the nanopore and results in a current blockade unique to each tag for sequence determination. The polymerase is covalently attached to the nanopore by a short linker so the polymeric tag will have sufficient time to enter the vestibule and constriction of the nanopore prior to its release ensuring that its current blockade signal is recorded by the nanopore. The extended DNA strand bears only natural nucleotides, enabling long reads. We have carried out the key proof-of-principle experiments to demonstrate the feasibility of this approach. Here our strong team of nucleic acid chemists, genomic scientists, electrical engineers, and nanofabrication experts will further develop the Nano-SBS as a high throughput genomic sequencing system. We will develop robust methodology to attach polymerase to the .-hemolysin (AHL) nanopore and synthesize nano-tags with unique chemical properties resulting in AHL current blockades distinct from each other and nucleotide precursors. We will test these elements in single pores as well as in new nanopore array chips with separate sensors and circuits for each pore. We will produce mutant AHL and polymerase constructs and link them to each other, selecting for the combination that assures accurate DNA extension reactions, and rapid capture and detection of tags in nanopores. The nanopore chips will be enhanced and expanded from the current 260 nanopores to over 125,000 using advanced nanofabrication techniques. We will conduct real-time single molecule Nano-SBS on DNA templates with known sequences to test and optimize the overall system. These research and development efforts will lay the foundation for the production of a commercial single molecule electronic DNA sequencing platform, which will enable routine use of sequencing for medical diagnostics and personalized medicine. 1

非常需要降低DNA测序的成本以实现1000美元的目标 基因组。我们最近通过合成（Nano-SB）开发了一种新的基于纳米孔的测序 方法。在这个项目中，我们将追求纳米SB的方法的开发 吞吐量实时单分子测序平台。在纳米SBS方法中，聚合物 尺寸和电荷的标签附着在四个的末端磷酸盐 核苷酸。当互补的核苷酸类似物进入模板 - 聚合物聚合酶时 在聚合酶反应期间附着在纳米孔上的复合物，特异性的标签 该核苷酸被捕获在纳米孔内的电压梯度中，并导致电流 每个标签独有的封锁以确定序列。聚合酶是共价附加的 通过短链接到纳米孔，因此聚合物标签将有足够的时间进入 纳米孔在释放之前的前庭和收缩，以确保其当前封锁 信号由纳米孔记录。扩展的DNA链只有天然核苷酸， 启用长阅读。我们已经进行了主要的原理实验证明 这种方法的可行性。在这里，我们强大的核酸化学家团队，基因组 科学家，电气工程师和纳米制造专家将进一步开发纳米SBS 作为高通量基因组测序系统。我们将开发出强大的方法论 将聚合酶连接到.-羟蛋白酶（AHL）纳米孔，并合成具有独特的纳米标签 化学特性导致AHL电流阻滞与彼此不同的核苷酸 前体。我们将在单个毛孔以及新的纳米孔阵列芯片中测试这些元素 每个孔都带有单独的传感器和电路。我们将产生突变的AHL和聚合酶 构造并互相链接，选择确保准确DNA的组合 扩展反应，以及纳米孔中标签的快速捕获和检测。纳米芯芯片 将增强并从当前的260纳米孔扩展到超过125,000 高级纳米制造技术。我们将对实时单分子纳米-SB进行 具有已知序列的DNA模板测试和优化整体系统。这些研究 开发工作将为生产商业单一的生产奠定基础 分子电子DNA测序平台，该平台将常规使用测序 医学诊断和个性化医学。 1