Optimization of Processive Enzymes for DNA Sequencing using Nanopores

使用纳米孔优化 DNA 测序的加工酶

基本信息

批准号：
8183739
负责人：
MARK A AKESON
金额：
$ 129.17万
依托单位：
UNIVERSITY OF CALIFORNIA SANTA CRUZ
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-08-15 至 2014-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8183739
关键词：
Area Back Biological California Centromere Collaborations Cytosine DNA DNA Sequence DNA amplification DNA biosynthesis DNA-Directed DNA Polymerase Detection Devices Engineering Environment Enzymes Epigenetic Process Excision Fluorescence Genetic Genome Genomics Goals Hemolysin Human Human Resources Individual Knowledge Laboratories Length Medicine Membrane Methods Methylation Modification Molecular Motion Motor Movement Noise Nucleotides Organism Pennsylvania Polymerase Population Predisposition Proteins Reading Research Resistance Resolution Saline Signal Transduction Single-Stranded DNA Sodium Chloride Solutions Speed Staging Structure Technology Testing Thick Time Universities Viral Washington Work active control akeson base cost detector dinitroaminophenol improved information gathering innovation meetings nanopore novel research study sensor silicon nitride solid state success trait viral DNA

项目摘要

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.

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 测序装置对长（最多 150 kb）单 DNA 链进行快速、高精度且廉价的测序。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 DNA 碱基之间的分辨率。该提案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）实验室迄今为止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和知识来实现四个具体目标。具体目标之一是利用 DNA 聚合酶 Phi29 扩展 UCSC 的有希望的结果，Phi29 是一种“分子步进电机”，能够通过纳米孔测序仪精确控制 DNA 运动。这项工作将利用现有人员和 UCSC 12 年在 α 溶血素蛋白纳米孔方面的成功经验，以扩展对纳米孔中 Phi29 DNA 聚合酶功能的理解。具体目标二是使用两个纳米孔来评估 Phi29 DNA 聚合酶功能，因为它们的碱基分辨率可能优于 α 溶血素纳米孔。第一个是 MspA，一种蛋白质纳米孔，华盛顿大学和 UCSC 团队将使用 Phi29 DNA 聚合酶对其进行评估。此次合作利用了 UCSC 使用 Phi29 DNAP 的专业知识和华盛顿大学 MspA 的专业知识。第二个纳米孔是具有荧光检测功能的固态超薄氮化硅孔，将由宾夕法尼亚大学（固态纳米孔制造商）和 UCSC 团队使用 Phi29 DNA 聚合酶进行评估。第三个具体目标是通过增加各个碱基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。目前 UCSC 的极端微生物专家将从耐盐生物体中分离出 DNA 聚合酶。具体目标 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。公共健康相关性：该提案开发了基于纳米孔的 DNA 测序，与现有技术相比，显着提高了 DNA 测序的速度和保真度。最终目标是足够的速度和成本降低，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遗传特征和倾向提供基础。 21 世纪的个性化医疗需要这种理解和技术。