Single Molecule Real Time Electronic Sequencing

单分子实时电子测序

• 批准号: 8545596
• 负责人: Serge Guy Lemay
• 金额: $ 127.75万
• 依托单位: COLUMBIA UNIV NEW YORK MORNINGSIDE
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-14 至 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8545596
• 关键词: Adsorption; Base Sequence; Buffers; Charge; Chemicals; Complex; Consumption; Copy Number Polymorphism; DNA; DNA Sequence; DNA-Directed DNA Polymerase; Deoxyribonucleotides; Detection; Development; Devices; Diagnosis; Electrodes; Electronics; Electrostatics; Elements; Engineering; Ensure; Fingerprint; Floor; Generations; Genetic Transcription; Genome; Geometry; Health; Ions; Label; Lead; Length; Libraries; Lighting; Link; Livestock; Malignant Neoplasms; Messenger RNA; Microfluidics; Molecular; Motion; Noise; Nucleotides; Optics; Oxidation-Reduction; Performance; Phase; Phosphoric Monoester Hydrolases; Photons; Physiological; Polymerase; Preparation; Property; Reaction; Reading; Reagent; Research; Route; Sampling; Signal Transduction; Spectrum Analysis; Surface; System; Techniques; Technology; Time; Transducers; analog; base; cost; cost effective; density; design; detector; disorder prevention; electric impedance; falls; food environment; genome sequencing; improved; inorganic phosphate; millisecond; nucleotide analog; pathogen; prototype; screening; sensor; signal processing; single molecule; solid state; technology development

DESCRIPTION (provided by applicant): Third-generation sequencing approaches are largely focusing on single-molecule strategies with the ability to achieve long read lengths. Single-molecule approaches require little or no sample preparation, saving time and reagent costs. They are more accurate since there is less chance of errors as no amplification is needed and there is no bias in molecular quantification. In addition, single-molecule techniques allow direct sequencing of mRNA, allowing understanding of post-transcription editing variations and copy-number studies. Ideally, single-molecule SBS can be massively-parallel and real-time, operating at synthesis rates as high as 1 msec for DNA polymerase, however complex optics required to collect photons efficiently make scaling of the platforms to high densities difficult. A promising route for overcoming the challenges to optical techniques is bioelectronic detection. The direct, real-time detection of this reaction product by electrical means represents a two-fold challenge. First, the minute amount of charge involved falls well below the noise floor for solid-state detection. Second, the presence of a high concentration of screening ions in physiological buffers greatly reduces the range and strength of electrostatic interactions. As a result, conventional electrical detection strategies, including impedance spectroscopy, field-effect detection and Faradaic reactions, lack sufficient sensitivity to detect single molecules. In this four-year effort, we develop a real-time, single-molecule sequencing approach based on the electrical detection of specifically engineered electrochemical tags that are attached to each of the four nucleotides. A base-specific electrochemical tag is released during the nucleotide incorporation; this tag is then activated through a phosphatase reaction to become redox active and is subsequently collected into a single molecule fingerprinting region (composed of four nanogap transducers). Redox cycling is used to produce an amplified signal for detection in the fingerprinting region. This approach to signal amplification is the electrical analog of fluorescen labels which see repeated excitation and emission under constant illumination to achieve detection gain. These nanogap transducers are integrated onto a CMOS integrated circuit in a highly multiplexed, parallel format. The proposed approach combines the advantages of single-molecule real time sequencing with a CMOS-compatible single molecule signal transduction platform and its attendant scalability benefits

描述（由申请人提供）：第三代测序方法主要集中在能够实现长读取长度的单分子策略上。单分子方法需要很少或不需要样品制备，节省了时间和试剂成本。它们更准确，因为不需要扩增，错误的可能性更小，并且分子定量中没有偏倚。此外，单分子技术允许对mRNA进行直接测序，允许理解转录后编辑变异和拷贝数研究。理想情况下，单分子SBS可以是并行的和实时的，对于DNA聚合酶以高达1毫秒的合成速率操作，然而有效收集光子所需的复杂光学器件使得平台难以缩放到高密度。 一个有前途 克服光学技术挑战的途径是生物电子检测。通过电子手段直接实时检测该反应产物代表了双重挑战。首先，所涉及的微小电荷量远低于固态检测的本底噪声，福尔斯。其次，生理缓冲液中高浓度屏蔽离子的存在大大降低了静电相互作用的范围和强度。因此，传统的电检测策略，包括阻抗谱，场效应检测和法拉第反应，缺乏足够的灵敏度来检测单个分子。 在这四年的努力中，我们开发了一种实时单分子测序方法，该方法基于对附着在四种核苷酸中的每一种上的专门设计的电化学标签的电检测。碱基特异性电化学标签在核苷酸掺入期间释放;然后该标签通过磷酸酶反应被激活以变得氧化还原活性，随后被收集到单分子指纹识别区域（由四个纳米间隙换能器组成）中。氧化还原循环用于产生放大的信号，用于指纹区域中的检测。这种信号放大的方法是荧光标记的电模拟，其在恒定照明下看到重复的激发和发射以实现检测增益。这些纳米间隙传感器以高度多路复用的并行格式集成到CMOS集成电路上。所提出的方法结合了单分子真实的时间测序与CMOS兼容的单分子信号转导平台的优点及其伴随的可扩展性益处