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：
8512765
负责人：
MARK A AKESON
金额：
$ 113.7万
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 2015-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8512765
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.

描述（由申请人提供）：该项目的长期目标是快速，高度准确且廉价的测序，对具有纳米孔的DNA测序设备的长（高达150 kb）的单个DNA链。达到这个长期目标需要精确控制DNA运动，经过纳米孔序列检测器，并改善DNA碱基之间的纳米孔序列检测器分辨率。该提案的具体目的是基于迄今为止在加利福尼亚大学圣克鲁斯分校（Akeson），华盛顿大学（Gundlach）和宾夕法尼亚大学（DRNDIC）实验室在这两个领域取得的进展。个人实验室专业知识和知识将被整合起来，以实现四个具体目标。特定的目标一使用DNA聚合酶PHI29在UCSC上扩展了有希望的结果，DNA聚合酶PHI29是一种能够通过纳米孔测序仪精确控制DNA运动的“分子步进运动”。这项工作将利用α-溶血素蛋白纳米孔在UCSC上运用现有人员和12年的成功，以扩展对纳米孔中PHI29 DNA聚合酶功能的理解。具体目标两个评估PHI29 DNA聚合酶功能的功能，其中两个纳米孔被选为其潜在的优质碱基分辨率，而不是α-溶血素纳米孔。第一个是MSPA，一种蛋白质纳米孔，将由华盛顿和UCSC团队U使用PHI29 DNA聚合酶进行评估。这种合作利用了UCSC的PHI29 DNAP以及华盛顿U的MSPA的专业知识来利用专业知识。第二种纳米孔是具有荧光检测的固态超薄硅孔，将通过penn（固态纳米孔的制造商）和UCSC团队的U使用PHI29 DNA聚合酶进行评估。第三个特定目标通过增加各个碱基的当前信号差异来改善DNA碱基分辨率。这将通过纳米孔中的盐浓度升高与使用耐盐性DNA聚合酶相结合来实现。来自UCSC目前的极端细胞专家将分离出耐盐生物的DNA聚合酶。特定目标4将使用收集的所有信息通过使用纳米孔测序设备对长（48 kb）DNA链进行测序来生成概念证明。这项技术的实现将为对人类和其他人群中个人遗传特征和倾向的个人遗传特征和倾向提供基础。