Sequencing by nanopore mass spectrometry

纳米孔质谱测序

• 批准号: 7714604
• 负责人: Derek Stein
• 金额: $ 27.69万
• 依托单位: BROWN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-01 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7714604
• 关键词: Affect; Biochemistry; Biological Sciences; Caliber; Charge; Cleaved cell; Coupled; Custom; DNA; DNA Fragmentation; DNA Sequence; Detection; Development; Electrons; Electrophoresis; Electrospray Ionization; Generations; Genetic; Genome; Genomics; Goals; Healthcare; Heart; Housing; Human Genome; Ions; Lasers; Liquid substance; Marriage; Mass Spectrum Analysis; Measurement; Membrane; Methods; Molecular; Nucleotides; Phase; Physiologic pulse; Process; Reading; Research; Sampling; Shapes; Solutions; Speed; System; Techniques; Technology; Testing; Travel; Vacuum; Variant; aqueous; base; detector; dimer; electric field; instrument; ionization; irradiation; mass spectrometer; nanopore; nanoscale; public health relevance; quantum; silicon nitride; single molecule; solid state; success

DESCRIPTION (provided by applicant): The goal of this project is to test the feasibility of a new single-molecule DNA sequencing strategy that combines solid-state nanopores with mass spectrometry. The idea is to sequentially cleave each nucleotide or base from a DNA molecule as it transits a nanopore, then identify each one by determining its mass-to-charge ratio in a mass spectrometer. Identifying the bases of a translocating DNA molecule by mass spectrometry is appealing because: 1) It is an extremely sensitive technique that can easily distinguish the four DNA bases from their significantly different masses; 2) Modern ion detectors can detect the impact of single ions with a quantum efficiency approaching unity; 3) Those same ion detectors register ions as ~ 20 ns electrical pulses, offering a high detection bandwidth that may obviate any need to control the DNA translocation speed; 4) The sequence of DNA is revealed by the order in which ions of different mass impact the detector, and is not affected by variations of translocation speed; 5) Mass measurements are expected to be insensitive to the orientation of a base in the nanopore, which is difficult to control. The success of our strategy hinges on whether ionized bases or nucleotides can be controllably cleaved from the leading end of a DNA molecule as it translocates the nanopore, and transferred into a mass spectrometer that is housed in a vacuum chamber. This project consequently focuses on assembling a nanopore mass spectrometry instrument, and using it to understand and control ionization and molecular fragmentation processes at the liquid-vacuum interface. The specific aims are to: 1) Detect DNA mononucleotides in a quadrupole mass spectrometer coupled to a ¿m-scale, chip-based pore; 2) Demonstrate mass spectrometry of single DNA bases ejected from a nanopore; 3) Identify efficient DNA fragmentation and ionization mechanisms; 4) Sequence short DNA homopolymers. Obtaining high quality sequence information (e.g. Q20 bases or better) from DNA homopolymers will demonstrate the viability of the nanopore mass spectrometry technique. This would justify developing a second-generation system, capable of sequencing long, heterogeneous DNA molecules. The $1000 per genome objective would be well within reach. Public Health Relevance: This project aims to develop a single-molecule DNA sequencing technology that would permit a full human genome to be sequenced for under $1000 and in under one day. This would have a profound impact on the life sciences by enabling genetic studies of exceptionally wide scope, and it would enable revolutionary health care options through personal genomics.

描述（由申请人提供）：该项目的目标是测试将固态纳米孔与质谱相结合的新单分子 DNA 测序策略的可行性，其想法是从 DNA 分子中顺序切割每个核苷酸或碱基。当它穿过纳米孔时，然后通过在质谱仪中确定其质荷比来识别每个碱基通过质谱法识别易位 DNA 分子的碱基很有吸引力，因为：1)这是一种极其灵敏的技术，可以轻松区分四种 DNA 碱基的质量显着不同；2) 现代离子检测器可以检测单个离子的影响，量子效率接近一致；3) 这些相同的离子检测器将离子记录为 ~ 20 ns 电脉冲，提供高检测带宽，可以消除控制 DNA 易位速度的任何需要；4) DNA 序列通过不同质量的离子撞击检测器的顺序来揭示，并且不受变化的影响5) 质量测量预计对纳米孔中碱基的方向不敏感，这很难控制，我们的策略的成功取决于电离碱基或核苷酸是否可以从纳米孔的前端可控地裂解。 DNA 分子在纳米孔中移位，并转移到真空室中的质谱仪中，因此该项目的重点是组装纳米孔质谱仪，并用它来理解和分析。控制液体-真空界面的电离和分子碎裂过程具体目标是：1) 在与 ¿ 耦合的四极质谱仪中检测 DNA 单核苷酸。 m 级、基于芯片的孔；2) 演示从纳米孔中喷射的单个 DNA 碱基的质谱分析；3) 识别有效的 DNA 断裂和电离机制；4) 获得高质量的序列信息（例如 Q20 碱基）。 DNA均聚物中的（更好）将证明纳米孔质谱技术的可行性，这将证明开发能够对长的、异质的测序的第二代系统是合理的。每个基因组 1000 美元的目标完全可以实现。 公共健康相关性：该项目旨在开发一种单分子 DNA 测序技术，能够以不到 1000 美元的价格在一天之内对完整的人类基因组进行测序。这将通过实现基因研究对生命科学产生深远的影响。范围异常广泛，它将通过个人基因组学实现革命性的医疗保健选择。