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.

我们提出了一种对单个DNA进行实时直接测序的方法 以天然DNA的速度和准确性从基因组DNA中提取分子 使用阴性核苷酸聚合。我们将利用真正的纳米- DNI复制中使用的机器，天然DNA聚合物。喜欢困难的你 工程师man-madu纳米结构usad在nanopore测序，以distingiish 4 碱基类型的紧密接近和恒定的絮凝，DNA聚合酶具有精确的 原子溶解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 低至百万分之一的碱基和高聚体，如phi-29 DNA 聚合酶一次可扩增多达10万个碱基。从...... 结构和动力学研究表明，DNA合成的保真度 基于精致的结构互补性和众多的具体 Pulymerose蛋白的活性位点与 引物/模板/核苷酸复合物。动态化学机械或构象 伴随特异性相互作用的变化，诱导拟合，键断裂/形成， 和模板易位确保高度准确和有序的碱基配对， 合并我们的策略是在聚合酶表面设计传感器， 蛋白质工程监测微小但明显的构象变化 伴随着每种碱基类型的掺入。一个小的距离变化（一个到 几十埃）可以用福斯特共振能量转换器精确测量 （FRET）技术。多个FRET paur或网络位于 聚合酶用于真实的时间（10倍）监测构象变化 比DNA合成的速率更快）。传感器将提供多参数 关于聚合酶的动态结构的信息，这很可能将提供 eech底座类型的enique签名。化学改性如 也可以潜在地检测到胫骨板DNA上的甲基化。有了这样的 方法，非常长的DNA分子可以在细节上高保真地测序， 人类基因组或甚至表观基因组可以在不到一小时内测序。在这 建议，我们的目标是调查是否有一个可区分的FRET信号 与四种不同核素的每一种通过DNA的结合有关 聚合体。