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学相结合。这个想法是当DNA分子通过纳米孔时，顺序地切割DNA分子中的每个核苷酸或碱基，然后通过在质谱仪中确定其质量/电荷比来识别每个核苷酸或碱基。用质谱仪鉴定DNA移位分子的碱基很有吸引力，因为：1)它是一种极其灵敏的技术，可以很容易地区分四种不同质量的DNA碱基；2)现代离子探测器可以探测单个离子的影响，量子效率接近1；3)相同的离子探测器将离子记录为~20 ns的电脉冲，提供了高检测带宽，可以避免任何控制DNA移位速度的需要；4)DNA的顺序是由不同质量的离子撞击探测器的顺序揭示的，不受移位速度的影响；5)质量测量应对纳米孔中碱基的取向不敏感，这很难控制。我们的策略的成功取决于，当DNA分子转移纳米孔时，电离碱基或核苷酸能否从DNA分子的前端可控地切割出来，并转移到安装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计，并使用它来了解和控制液-真空界面的电离和分子碎裂过程。其具体目标是：1)在四极质谱计中检测DNA单核苷酸，该质谱仪连接到一个m尺度的芯片基孔；2)展示从纳米孔中喷出的单个DNA碱基的质谱学；3)识别有效的DNA碎裂和电离机制；4)序列短DNA均聚物。从DNA均聚体获得高质量的序列信息(例如Q20碱基或更高)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、不同种类的DNA分子进行测序。每个基因组1000美元的目标将是遥不可及的。 与公共健康相关：该项目旨在开发一种单分子DNA测序技术，该技术将允许以低于1000美元的价格对完整的人类基因组进行测序，并在一天内完成。这将对生命科学产生深远的影响，因为它将使非常广泛的基因研究成为可能，并将通过个人基因组学实现革命性的医疗保健选择。

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.