Optimization of Processive Enzymes for DNA Sequencing using Nanopores

描述（由申请人提供）：该项目的长期目标是使用基于纳米孔的DNA测序设备对长（高达150kb）的单DNA链进行快速，高度准确和廉价的测序。实现这一长期目标需要精确控制DNA通过纳米孔序列检测器的运动，并提高纳米孔序列检测器在DNA碱基之间的分辨率。这项建议的具体目标建立在加州大学圣克鲁斯分校（Akeson）、华盛顿大学（Gundlach）和宾夕法尼亚大学（Drndic）的实验室迄今在这两个领域取得的进展的基础上。个别实验室的专业知识和知识将整合，以实现四个具体目标。具体目标之一是扩展UCSC的DNA聚合酶Phi29的有希望的结果，这是一种能够通过纳米孔测序仪精确控制DNA运动的“分子步进马达”。这项工作将利用现有人员和加州大学圣地亚哥分校在α溶血素蛋白纳米孔方面12年的成功经验，扩展对纳米孔中Phi29 DNA聚合酶功能的理解。特异性目标2评估Phi29 DNA聚合酶的功能，选择两个纳米孔，因为它们比α溶血素纳米孔具有潜在的更好的碱基分辨率。第一种是蛋白质纳米孔MspA，华盛顿大学和UCSC的团队将用Phi29 DNA聚合酶对其进行评估。此次合作充分利用了UCSC的Phi29 DNAP和华盛顿大学MspA的专业知识。第二个纳米孔是具有荧光检测功能的固态超薄氮化硅孔，宾州大学（固态纳米孔的制造商）和加州大学圣地亚哥分校的团队将用Phi29 DNA聚合酶对其进行评估。第三个具体目标是通过增加来自单个碱基的电流信号的差异来提高DNA碱基分辨率。这将通过增加纳米孔中的盐浓度并结合使用耐盐DNA聚合酶来实现。来自耐盐生物的DNA聚合酶将由目前在UCSC的极端微生物专家分离。Specific aim 4将使用收集到的所有信息，通过使用纳米孔测序设备对长（最多48 KB） DNA链进行测序，来生成概念证明。这项技术的实现将为更全面地了解人类和其他人群的个体遗传特征和倾向提供基础。

DESCRIPTION (provided by applicant): The long-term objective of this project is rapid, highly accurate, and inexpensive sequencing of long (up to 150 kb) single DNA strands with a nanopore based DNA sequencing device. Meeting this long-term objective requires precise control of DNA movement past a nanopore sequence detector, and improvement of nanopore sequence detector resolution between DNA bases. The specific aims of this proposal build upon progress made to date in these two areas by laboratories at the University of California, Santa Cruz (Akeson), University of Washington (Gundlach) and the University of Pennsylvania (Drndic). Individual laboratory expertise and knowledge will be integrated to accomplish four specific aims. Specific aim one extends promising results at UCSC with DNA polymerase Phi29, a "molecular step motor" able to precisely control DNA movement through a nanopore sequencer. This work will employ existing personnel and 12 years of success at UCSC with alpha hemolysin protein nanopores to extend understanding of Phi29 DNA polymerase function in a nanopore. Specific aim two evaluates Phi29 DNA polymerase function with two nanopores selected for their potentially superior base resolution to the alpha hemolysin nanopore. The first is MspA, a protein nanopore, which will be evaluated with Phi29 DNA polymerase by U of Washington and UCSC teams. This collaboration takes advantage of expertise with use of Phi29 DNAP at UCSC and expertise with MspA at U of Washington. The second nanopore, a solid state ultrathin silicon nitride pore with fluorescence detection, will be evaluated with Phi29 DNA polymerase by U of Penn (makers of the solid-state nanopore) and UCSC teams. The third specific aim improves DNA base resolution through increased differences in current signals from individual bases. This will be achieved by increased salt concentrations in the nanopore combined with use of salt tolerant DNA Polymerases. DNA polymerases from salt tolerant organisms will be isolated by extremophile experts currently at UCSC. Specific aim 4 will use all information gathered to generate proof of concept through sequencing of long (up to 48 KB) DNA strands using a nanopore sequencing device. Realization of this technology will provide the basis for a more complete understanding of individual genetic traits and predispositions in human and other populations.