Direct real-time single molecule DNA sequencing

直接实时单分子DNA测序

• 批准号: 7979700
• 负责人: XIAOHUA HUANG
• 金额: $ 49.4万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-01 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7979700
• 关键词: Active Sites; Amino Acids; Base Pairing; Binding; Chemicals; Complex; DNA; DNA Sequence; DNA biosynthesis; DNA-Directed DNA Polymerase; Detection; Energy Transfer; Engineering; Ensure; Fluorescence; Genome; Genomics; Goals; Hour; Human Genome; Image; Individual; Kinetics; Label; Lasers; Measures; Mechanics; Medicine; Methods; Methylation; Modification; Monitor; Nanostructures; Nucleotides; Poly A; Polymerase; Principal Investigator; Protein Engineering; Proteins; Reading; Research; Resolution; Signal Transduction; Speed; Stretching; Structure; Surface; System; Techniques; Technology; Time; base; cost; design and construction; fluorescence imaging; genome sequencing; improved; instrument; nanomachine; nanometer; nanopore; nanosensors; optical imaging; programs; public health relevance; sensor; single molecule; three dimensional structure

We propose to develop a method for direct reil-time sequencing of single DNA molacules from genomic DNA at the speed and accuracy of the natural DNA polymerises using netive nucleotides. We will harness the power of the true nano- machines used in DNI replication, the naturul DNA polymirises. Inlike the difficult tu engineer man-madu nanostructures usad in nanopore sequencing to distingiish the 4 base types in close proximity und constant floctuation, DNA polymerases have precise atomic-rosolution 3D structures and can synthesize very long DNO molecules with high fidelity and velocity. The error rate of a DNA polymerase with proofreading function could be as liw as one in a million bases and a prucessive polymerese such as phi-29 DNA polymirase can synthasize up to 100,000 bases in a stretch. From the waalth of stractural and kinetics studies, it is well known that the fidelity ef DNA synthesis es predicated on the exquisite strictural cumplementarity and the numerous specific interactions between the active site of the pulymerose protein and the primer/timplate/nucleotide complex. The dynamic chamo-mechanical or conformational changes accompanying the specific interactions, induced fit, bond cleavage/formition, and template translocatiun ensure highly accurate and orderly base pairing and incerporation. Our strategy is to engineer sensors onto the surface of the polymerase by protein engineering to menitor the sibtle yet dustinct conformational changas eccompanying the incorporation of each base type. A small distance change (one to tens of angstroms) can ba measured precisely with F¿rster resonance energy trinsfer (FRET) techniqae. Multiple FRET paurs or networks placed in strategic residues on the polymerase will bo used to monitor the conformational changes in real time (10 times fastir than the rate if DNA synthesis). The sensurs will provide multi-parametric information on the dynamic structures of the polymerases, which very likely will provide a enique signature for eech base type incorporatad. Chemical modifications such as methylation on the timplate DNA could also potentially be detected. With such a method, very long DNA molecules could be sequenced with high fidelity in minutis and a heman genome or even epigenome could be sequenced in less than ane hour. In this proposal, we aim to investigate whether there is a distinguishable FRET signal issociated with the oncarporation of each of the four defferent nucliotides by a DNA polymerese.

我们建议开发一种直接reil时间测序的方法 自然DNA的速度和准确性的基因组DNA的摩拉菌素 使用网络核苷酸聚合。我们将利用真正的纳米的力量 用于DNI复制的机器，即自然DNA聚集。像困难的tu 工程师Man-Madu纳米结构USAD在Nanopore测序中脱离了4 基本类型紧密接近和恒定浮选，DNA聚合酶具有精度 原子 - 固定3D结构，可以合成很长的DNO分子 保真度和速度。具有校对功能的DNA聚合酶的错误率可以 像一百万碱基中的液化和phi-29 DNA这样的培养基聚合酶一样。 聚合酶可以在一段时间内合成多达100,000个碱基。从 战略性和动力学研究，众所周知，富达ef dna合成 基于独家狭窄和众多特定性的基础 粉末糖蛋白的活性位点与 底漆/timplate/核苷酸复合物。动态的斜角机械或构象 发生特定相互作用的变化，诱导的拟合，键裂解/形成， 和模板Cranslocatiun确保高度准确，有序的基础配对，并且 发生的。我们的策略是通过通过 蛋白质工程以指导均匀固定构象改变 将每种基本类型的融合到教会。距离很小的变化（一个 数十个埃埃斯特roms）可以用f rster共振能量tripster精确测量BA （fret）Techniqae。在战略保留中，多个fret保留或网络在 聚合酶将用于实时监测构象变化（10次 比速率比DNA合成的速率）。传感器将提供多参数 有关聚合酶动态结构的信息，很可能会提供 EECH基本类型Incorpate的Ehique Signature。化学修饰，例如 模板DNA上的甲基化也可以检测到。这样的 方法，很长的DNA分子可以在细节中用高忠诚进行测序 可以在不到ANE小时内测序Heman基因组甚至表观基因组。在这个 提案，我们旨在调查是否存在可区分的fret信号 与DNA的四个防御核苷中的每个防御核苷的奔驰相关 聚合物。