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150 kb)的单链DNA进行快速、高精度和廉价的测序。要实现这一长期目标，需要精确控制通过纳米孔序列检测器的DNA移动，并提高DNA碱基之间的纳米孔序列检测器的分辨率。这项提案的具体目标建立在加州大学圣克鲁斯分校(阿克森)、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。将整合各个实验室的专业知识，以实现四个具体目标。具体目标一利用DNA聚合酶Phi29在UCSC推广了令人振奋的成果，Phi29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”。这项工作将利用加州大学洛杉矶分校现有的人员和在阿尔法溶血素蛋白纳米孔12年的成功经验来扩大对纳米孔中Phi29 DNA聚合酶功能的理解。具体目的二评估Phi29 DNA聚合酶的功能，选择两个纳孔，因为它们可能比α溶血素纳米孔具有更高的碱基分辨率。第一个是MSPA，一种蛋白质纳米孔，华盛顿大学和加州大学旧金山分校的团队将用Phi29 DNA聚合酶对其进行评估。这次合作利用了加州大学洛杉矶分校使用Phi29 DNAP的专业知识和华盛顿大学MSPA的专业知识。第二个纳米孔是固态超薄氮化硅孔，具有荧光检测功能，将由宾夕法尼亚大学(固态纳米孔制造商)和UCSC团队用Phi29 DNA聚合酶进行评估。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碱基的分辨率。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聚合酶将由目前在加州大学圣迭戈分校的极端细菌专家分离。具体目标4将使用收集的所有信息，通过使用纳米孔测序设备对长(最多48kb)DNA链进行测序来产生概念证据。这项技术的实现将为更全面地了解人类和其他群体中的个体遗传特征和易感性提供基础。 公共卫生相关性：这项提案开发了基于纳米孔的DNA测序，大大提高了DNA测序的速度和保真度，超过了当前的技术。最终的目标是充分的速度和成本的降低，以允许对个体基因组进行常规测序，并最终为详细了解个体的遗传特征和易感性提供基础。这种理解和技术是21世纪个性化医学所需要的。

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. PUBLIC HEALTH RELEVANCE: This proposal develops nanopore based DNA sequencing for significant improvement in speed and fidelity of DNA sequencing over current technologies. The ultimate goal is sufficient speed and cost reduction to permit routine sequencing of individual genomes and ultimately provide the basis for a detailed understanding of individual genetic traits and predispositions. This understanding and technology are needed for personalized medicine in the 21st century.