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Single Molecule Real Time Electronic Sequencing

单分子实时电子测序

基本信息

批准号：
8929279
负责人：
Serge Guy Lemay
金额：
$ 123.63万
依托单位：
COLUMBIA UNIV NEW YORK MORNINGSIDE
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-09-14 至 2019-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8929279
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

项目摘要

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关注能够实现长读长的单分子策略。单分子方法需要很少或不需要样品制备，从而节省时间和试剂成本。它们更加准确，因为错误的可能性较小，因为不需要扩增并且分子定量中不存在偏差。此外，单分子技术可以直接对 mRNA 进行测序，从而可以了解转录后编辑变异和拷贝数研究。理想情况下，单分子 SBS 可以大规模并行和实时运行，DNA 聚合酶的合成速率高达 1 毫秒，但有效收集光子所需的复杂光学器件使得平台难以扩展到高密度。一个有前途的克服光学技术挑战的途径是生物电子检测。通过电学手段直接、实时检测这种反应产物面临着双重挑战。首先，所涉及的微量电荷远低于固态检测的本底噪声。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个核苷酸中的每一个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过程中被释放；然后，该标签通过磷酸酶反应被激活，变得具有氧化还原活性，并随后被收集到单分子指纹识别区域（由四个纳米间隙传感器组成）。氧化还原循环用于产生放大信号，用于指纹识别区域的检测。这种信号放大方法是荧光标记的电模拟，荧光标记在恒定照明下重复激发和发射，以实现检测增益。这些纳米间隙传感器以高度多路复用、并行的形式集成到 CMOS 集成电路上。所提出的方法结合了单分子实时测序的优点和 CMOS 兼容的单分子信号转导平台及其随之而来的可扩展性优势