Sequencing by nanopore mass spectrometry

描述（由申请人提供）：该项目的目标是测试一种新的单分子DNA测序策略的可行性，该策略将固态纳米孔与质谱法相结合。这个想法是在DNA分子通过纳米孔时依次切割每个核苷酸或碱基，然后通过在质谱仪中确定其质荷比来识别每一个。通过质谱法鉴定易位DNA分子的碱基是有吸引力的，因为：1）它是一种非常灵敏的技术，可以容易地从它们显著不同的质量中区分四种DNA碱基; 2）现代离子检测器可以检测具有接近1的量子效率的单个离子的影响; 3）这些相同的离子检测器将离子记录为~ 20 ns的电脉冲，提供了高检测带宽，其可以消除控制DNA移位速度的任何需要; 4）DNA的序列通过不同质量的离子撞击检测器的顺序来揭示，并且不受移位速度变化的影响; 5）预期质量测量对纳米孔中碱基的取向不敏感，这是难以控制的。我们的策略的成功取决于电离的碱基或核苷酸是否可以在DNA分子易位纳米孔时从DNA分子的前端可控地切割，并转移到容纳在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，并使用它来理解和控制液体-真空界面处的电离和分子碎片化过程。具体目标是：1）在耦合到μ m级基于芯片的孔的四极质谱仪中检测DNA单核苷酸; 2）演示从纳米孔喷射的单个DNA碱基的质谱; 3）识别有效的DNA片段化和电离机制; 4）序列短DNA均聚物。从DNA均聚物获得高质量的序列信息（例如Q20碱基或更好）将证明纳米孔质谱技术的可行性。这将证明开发第二代系统是合理的，该系统能够对长的异质DNA分子进行测序。每个基因组1000美元的目标将完全可以实现。 公共卫生相关性：该项目旨在开发一种单分子DNA测序技术，该技术将允许在不到1000美元和不到一天的时间内对完整的人类基因组进行测序。这将对生命科学产生深远的影响，使遗传研究的范围非常广泛，它将使革命性的医疗保健选择，通过个人基因组学。

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.