Single Molecule Real Time Electronic Sequencing

描述(由申请人提供)：第三代测序方法主要关注具有实现长阅读长度的能力的单分子策略。单分子方法需要很少或根本不需要样品制备，从而节省了时间和试剂成本。它们更准确，因为不需要扩增，而且在分子定量中没有偏差，所以出错的可能性更小。此外，单分子技术允许对mRNA进行直接测序，从而能够理解转录后编辑变异和拷贝数研究。理想情况下，单分子SBS可以是大规模并行和实时的，对于DNA聚合酶来说，其合成速度高达1毫秒，然而，高效收集光子所需的复杂光学系统使平台难以扩展到高密度。一个有希望的人 克服光学技术挑战的途径是生物电子检测。通过电气手段直接、实时地检测这种反应产物是一项双重挑战。首先，所涉及的微小电荷量远低于固态检测的噪声下限。其次，生理缓冲液中存在高浓度的屏蔽离子，极大地降低了静电相互作用的范围和强度。因此，传统的电学检测方法，包括阻抗光谱、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的单分子测序方法，该方法基于对贴在四个核苷酸上的特定工程的电化学标签的电子检测。碱基特异的电化学标签在核苷酸掺入过程中释放；然后通过磷酸酶反应激活该标签，使其成为氧化还原活性，并随后被收集到单分子指纹区域(由四个NanoGap传感器组成)。氧化还原循环被用来产生放大的信号，用于在指纹区进行检测。这种信号放大的方法是荧光标记的电气模拟，它在恒定的光照下看到重复的激发和发射，以实现检测增益。这些NanoGap传感器以高度多路复用、并行的格式集成到一块cmos集成电路上。该方法结合了单分子实时测序和与cmos兼容的单分子信号转导平台的优点以及随之而来的可扩展性优势。

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