An Integrated System for Single Molecule Electronic Sequencing by Synthesis

有一个很大的需要，以减少DNA测序的成本，以实现1000美元的目标， 基因组我们最近开发了一种新的基于纳米孔的合成测序（Nano-SBS） approach.在这个项目中，我们将追求纳米SBS方法的发展， 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中的每一个的末端磷酸盐上。 个核苷酸当互补核苷酸类似物进入模板-引物-聚合酶 在聚合酶反应期间附着到纳米孔的复合物中，特异性标记 该核苷酸在纳米孔内的电压梯度中被捕获 每个标签都具有独特的阻断，用于序列测定。聚合酶共价连接在 因此聚合物标签将有足够的时间进入纳米孔。 纳米孔的前庭和收缩在其释放之前确保其电流阻断 信号由纳米孔记录。延伸的DNA链仅带有天然核苷酸， 允许长读取。我们已经进行了关键的原理验证实验来证明 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、电子工程师和纳米纤维专家将进一步开发纳米SBS 作为高通量基因组测序系统。我们将制定强有力的方法， 将聚合酶连接到溶血素（阿勒）纳米孔和合成具有独特 化学性质导致阿勒电流阻断彼此不同， 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 每个孔都有独立的传感器和电路。我们将生产突变的阿勒和聚合酶 构建并将它们彼此连接，选择组合以确保准确的DNA 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个纳米孔增强和扩展到超过125，000个， 先进的纳米纤维技术我们将进行实时单分子纳米SBS上 使用已知序列的DNA模板来测试和优化整个系统。这些研究 和开发工作将奠定基础，生产一个商业单一的 分子电子DNA测序平台，这将使测序的常规使用， 医疗诊断和个性化医疗。 1

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