Characterization and Sequencing of Individual Polynucleotides via Solid State Ion Channels

项目摘要我们寻求一种新的技术，用于对DNA等多核苷酸进行超高速表征和测序。该方法依赖于这样的概念，即线性聚合物通过不渗透界面中适当小的通道或孔，将以反映聚合物中单体序列的方式顺序地改变孔的局部性质。我们最近使用模型RNA和DNA聚合物的结果证明了这种方法的前景，这些聚合物是通过脂质双层中的蛋白质通道进行电泳驱动的，但受到有机蛋白质-脂肪界面的微妙限制，该界面不能容忍变性剂和消除天然多核苷酸中碱基配对和其他二级结构所需的温度。这项提议的短期目标是开发一种坚固的无机界面(氮化硅)，它将复制有机界面所获得的结果，并导致开发一种用于极高速测定天然多核苷酸的仪器。实现这些短期目标将证明利用无机界面上的小通道作为开发新型传感设备用于单DNA分子高速测序的可行性。由于这种方法消除了DNA测序中目前所需的几个准备化学步骤，而且它依赖于固有的快速分子事件，因此这种方法的成功将是一项非常重要的进步，应该会将核酸测序的时间和成本减少几个数量级。

PROJECT SUMMARY We seek a new technique for very high speed characterization and sequencing of polynucleotides such as DNA. The approach relies on the notion that a linear polymer passing through a suitably small channel or pore in an otherwise impermeable interface will sequentially modify the local properties of the pore in a manner that reflects the sequence of monomers in the polymer. Our recent results using model RNA and DNA polymers that were electrophoretically driven through a protein channel in a lipid bilayer demonstrate the promise of this approach, but are limited by the delicacy of the organic protein-lipid interface which cannot tolerate the denaturants and temperatures required to eliminate base pairing and other secondary structures in native polynucleotides. The short term goals of this proposal are to develop a robust, inorganic interface (silicon nitride) that will duplicate the results obtained with the organic interface and lead to the development of an instrument for very high speed sizing of native polynucleotides. Accomplishing these short term goals will demonstrate the feasibility of using a small channel in an inorganic interface as the base on which to develop a novel sensing device for high speed sequencing of single DNA molecules. Because this approach eliminates several of the currently required preparatory chemical steps in DNA sequencing, and because it depends on inherently rapid, molecular events, success with this method will be an extraordinarily important advance that should reduce the time and cost of nucleic acid sequencing by several orders of magnitude.