DNA SEQUENCING BY LASER BASED MASS SPECTROMETRY

这项提案的重点是开发一种高速DNA测序系统 基于对桑格二脱氧测序产物的质谱分析。这个 目前的实验目标是对1000条模板DNA链进行测序 速率超过10兆基数/天的原始序列的基本范围 机器。测序方法涉及到质谱学(气相) 分离检测方法。为了实现这一目标，目前 酶DNA测序策略将与三个新的 生产一种低成本、易于自动化的测序方法的技术。 激光蒸发被用来将标记的DNA链转移到气体中 相位。一旦在气相共振增强，多光子电离是 用于在每一股上精确地放置一个单位的正电荷 然后进行飞行时间质量分析。所需的时间 获得能够分析所有测序的单一质谱图 产品不到L毫秒。这样的分析率将 加快测序速度，远远超过每天1兆数据库。巴赫 分离和检测过程以及由此产生的数据 文件，很容易实现自动化。这种方法旨在取代凝胶 目前用于分析DNA产物的电泳法 对反应进行排序。我们提议的技术的自动化将导致 在一种非常敏感的DNA测序方法中，只需要很少的人工操作 劳力。使用这种方法的仪器所产生的测序成本 方法应低于0.10美元的每个序列碱基，并可以 每个基数可能不到1美分。因为我们和其他人已经 展示了解吸长度超过500个碱基的DNA的能力， 这项提案中的大部分工作都涉及气体的冷却 阶段DNA和发展的光电离过程。为了增强 我们正在研究新的薄膜系统和集成 脉冲喷嘴进入我们现有的两个激光汽化/激光电离 用于研究DNA的飞行时间质谱学系统 测序。为了加强光电离过程，我们正在研究新的 寡核苷酸的电离标记，以及研究新的 电离方案。电离步骤有可能提供 高质量谱分辨率(与长读取长度相关) 模板链)和与其他质谱学相比的灵敏度 方法：研究方法。

This proposal focuses on developing a high speed DNA sequencing system based on mass spectral analysis of Sanger dideoxy sequencing products. The current experimental goal is to sequence template DNA strands in the 1,000 base range with rates exceeding 10 megabase of raw sequence per day per machine. The sequencing method involves a mass spectroscopic (gas phase) separation and detection method. To achieve this objective, current enzymatic DNA sequencing strategies will be combined with three new technologies to produce a low-cost, easily automated sequencing method. Laser vaporization is used to transfer tagged strands of DNA into the gas phase. Once in the gas phase resonance-enhanced, multiphoton ionization is used to place precisely one unit of positive charge on each strand and then time-of-flight mass analysis is performed. The time required to obtain a single mass spectrum capable of analyzing all of the sequencing products is less than l millisecond. Such an analysis rate would accelerate the sequencing rate well in excess of 1 megabase per day. Bach of the separation and detection processes, as well as the resulting data files, are readily automated. The method is designed to replace the gel electrophoretic methods currently used to analyze the products of DNA sequencing reactions. Automation of the technology we propose would result in an extremely sensitive DNA sequencing method requiring little manual labor. The sequencing cost resulting from an instrument that uses this methodology should be less than $0.10 per sequenced base, and could potentially be less than 1 cent per base. Since we and others have already demonstrated the ability to desorb DNA longer than 500 bases in length, the majority of the work in this proposal concerns both cooling of the gas phase DNA and developing the photoionization process. To enhance the cooling we are studying new thin film systems and the integration of a pulsed nozzle into our two existing laser vaporization/laser ionization time-of-flight mass spectroscopic systems dedicated to studying DNA sequencing. To enhance the photoionization process we are studying new ionization labels for oligonucleotides, as well as investigating new ionization schemes. The ionization step has the potential of providing high mass spectral resolution (correlating to long read lengths on the template strands) and sensitivity in comparison with other mass spectral methods.