An Integrated System for Single Molecule Electronic Sequencing by Synthesis

单分子电子合成测序集成系统

基本信息

批准号：
8572847
负责人：
GEORGE M CHURCH
金额：
$ 175万
依托单位：
COLUMBIA UNIV NEW YORK MORNINGSIDE
依托单位国家：
美国
项目类别：
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/8572847
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

项目摘要

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

为了实现 1000 美元的目标，迫切需要降低 DNA 测序的成本基因组。我们最近开发了一种新的基于纳米孔的边合成边测序（Nano-SBS）方法。在这个项目中，我们将致力于将纳米 SBS 方法发展为高高通量实时单分子测序平台。在纳米 SBS 方法中，聚合物不同大小和电荷的标签附着在四个的末端磷酸盐上核苷酸。当互补核苷酸类似物进入模板引物聚合酶时在聚合酶反应过程中附着在纳米孔上的复合物，该标签特异于核苷酸被纳米孔内的电压梯度捕获并产生电流每个标签独有的封锁用于序列确定。聚合酶是共价连接的通过短连接体连接到纳米孔，因此聚合物标签将有足够的时间进入纳米孔释放前的前庭和收缩确保其当前的封锁信号由纳米孔记录。延伸的 DNA 链仅含有天然核苷酸，启用长读取。我们进行了关键的原理验证实验来证明该方法的可行性。这里是我们强大的核酸化学家、基因组团队科学家、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。我们将开发稳健的方法将聚合酶附着到.-溶血素 (AHL) 纳米孔上并合成具有独特的纳米标签导致 AHL 当前阻断彼此不同且核苷酸不同的化学特性前体。我们将在单孔以及新的纳米孔阵列芯片中测试这些元素每个孔都有单独的传感器和电路。我们将生产突变型AHL和聚合酶构建并将它们相互连接，选择确保准确 DNA 的组合延伸反应以及纳米孔中标签的快速捕获和检测。纳米孔芯片将从目前的 260 个纳米孔增强并扩展到超过 125,000 个先进的纳米制造技术。我们将进行实时单分子纳米SBS 具有已知序列的 DNA 模板，用于测试和优化整个系统。这些研究开发工作将为商业单曲的制作奠定基础分子电子DNA测序平台，这将使测序的常规使用成为可能医疗诊断和个性化医疗。 1