DNA SEQUENCING BY LASER BASED MASS SPECTROMETRY

通过激光质谱法进行 DNA 测序

• 批准号: 2208866
• 负责人: ROBERT J LEVIS
• 金额: $ 15.53万
• 依托单位: WAYNE STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-09-30 至 1996-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2208866
• 关键词: DNA; biomedical automation; genetic techniques; health economics; ionization; laser spectrometry; mass spectrometry; method development; nucleic acid sequence

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.

该方案的重点是开发一种高速DNA测序系统 基于桑格双脱氧测序产物的质谱分析。的 目前的实验目标是对1000个DNA链中的模板DNA链进行测序， 基本范围，速率超过10兆字节的原始序列/天， 机测序方法涉及质谱（气相） 分离检测方法。为了实现这一目标，目前 酶促DNA测序策略将与三种新的 技术，以产生低成本，易于自动化测序方法。 激光蒸发用于将标记的DNA链转移到气体中 相位一旦在气相共振增强，多光子电离， 用于在每条链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 产品小于1毫秒。这样的分析率将 将测序速度大大加快，超过每天1兆碱基。巴赫 分离和检测过程以及由此产生的数据 文件，自动化。该方法旨在取代凝胶 目前用于分析DNA产物的电泳方法 测序反应我们提出的技术的自动化将导致 在一个极其灵敏的DNA测序方法中， 劳动使用这种仪器的测序成本 每个测序碱基的成本应低于0.10美元， 可能低于每个基数1美分。因为我们和其他人已经 证明了解吸长度超过500个碱基的DNA的能力， 该方案中的大部分工作涉及气体的冷却， 阶段DNA和发展光电离过程。增强 冷却，我们正在研究新的薄膜系统和集成的 脉冲喷嘴到我们现有的两个激光汽化/激光电离 专门用于研究DNA的飞行时间质谱系统 测序为了增强光电离过程，我们正在研究新的 寡核苷酸的电离标记，以及研究新的 电离方案电离步骤具有提供 高质谱分辨率（与质谱仪上的长读取长度相关） 模板链）和灵敏度与其他质谱 方法.