Real-time single-molecule nucleic acid sequencing with fluorogenic nucleotides

使用荧光核苷酸进行实时单分子核酸测序

• 批准号: 8136799
• 负责人: XIAOLIANG SUNNEY XIE
• 金额: $ 51.6万
• 依托单位: HARVARD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-17 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8136799
• 关键词: Animal Model; Base Sequence; Buffers; Cell Culture Techniques; Cells; Color; Computer Systems; DNA; DNA Sequence; DNA-Directed DNA Polymerase; Data; Detection; Devices; Digestion; Diploidy; Dyes; Ensure; Enzymatic Biochemistry; Fiber; Fluorescence; Generations; Genetic Materials; Genome; Genomics; Goals; Human Genome; Image; Individual; Label; Lasers; Lead; Length; Libraries; Medicine; Methods; Microfluidic Microchips; Microfluidics; Monitor; Nucleic Acids; Nucleic acid sequencing; Nucleotides; Phosphoric Monoester Hydrolases; Polymerase; Polyphosphates; Preparation; Process; Reaction; Reading; Reagent; Running; Sampling; System; Techniques; Technology; Time; abstracting; base; charge coupled device camera; cost; density; experience; fluorescence microscope; fluorophore; genome sequencing; inorganic phosphate; lithography; nano; novel; novel strategies; quantum; seal; single molecule

Real-time single-molecule nucleic acid sequencing with fluorogenic nucleotides Project Summary/Abstract Capitalizing on our group's experience on single molecule enzymology, we propose a novel method for multiplex sequencing of individual nucleic acid molecules using a sequencing-by-synthesis approach that employs fluorogenic nucleotide substrates. Upon incorporation of a non-fluorescent, terminal phosphate-labeled nucleotide substrate by a polymerase, a fluorogenic polyphosphate molecule is released, and subject to fast enzymatic digestion, yielding a single fluorophore, the color of which is dependent on the identity of the incorporated nucleotide. To facilitate single molecule fluorescence detection, an individual nucleic acid molecule is confined in a sealed sub-femtoliter nanoreactor, in which the sequencing reaction takes place continuously. Using conventional soft lithography, we fabricate an array of nanoreactors that allow simultaneous, real-time monitoring of thousands of isolated sequencing reactions with a fluorescence microscope and CCD camera. Our new approach offers low reagent cost, long read lengths, easy sample preparation, and high throughput at several megabases per minute. We also propose the integration of a massively parallel single molecule fluorogenic sequencer with microfluidic devices that process and deliver genetic material from a single cell.

荧光核苷酸实时单分子核酸测序 项目总结/摘要 利用我们小组在单分子酶学方面的经验，我们提出了一种新的方法， 对于使用合成测序对单个核酸分子进行多重测序， 使用荧光核苷酸底物的方法。在掺入非荧光剂后， 聚合酶末端磷酸标记核苷酸底物，荧光多磷酸 分子被释放，并进行快速酶消化，产生单一的荧光团，颜色 其依赖于掺入的核苷酸的特性。为了促进单个分子 在荧光检测中，单个核酸分子被限制在密封的亚飞升中， 纳米反应器，其中测序反应连续发生。使用传统的软 光刻，我们制造了一个纳米反应器阵列，允许同时，实时监测 成千上万的分离测序反应与荧光显微镜和CCD相机。我们 新方法提供了低试剂成本、长读取长度、易于样品制备和高 每分钟几兆字节的吞吐量。我们还提出了一个大规模并行的集成 具有微流体装置的单分子荧光测序仪 一个细胞的物质。