SEQUENCING OF DNA BY LASER IONIZATION

通过激光电离进行 DNA 测序

• 批准号: 2208594
• 负责人: CHRISTOPHER H BECKER
• 金额: $ 35.85万
• 依托单位: SRI INTERNATIONAL
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-08-01 至 1996-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2208594
• 关键词: DNA; biomedical equipment development; biotechnology; gas chromatography mass spectrometry; ionization; laser spectrometry; mass spectrometry; nucleic acid sequence

The primary aim of this research is to develop laser desorption time-of- flight mass spectrometry into a practical, very high speed, DNA sequencing technology as a direct replacement for gel electrophoresis. The mass spectrometer will measure the parent molecular masses from nested sets of single-stranded DNA oligomers prepared by a Sanger-type chemistry. The potential orders-of-magnitude increase in sequencing speed, and lowering of costs per base, derives from the inherently fast mass separation performed in the time-of-flight mass spectrometer. The experimental arrangement involves production of molecular ions by irradiating with a single pulsed laser beam the single-stranded DNA oligomer mixtures that are dispersed within a host chemical matrix. The natures of the matrix and the laser-induced desorption are central to the research. Major progress toward the project's technical goals has been accomplished, especially with our discovery of a particularly effective new ultraviolet (UV) light absorbing matrix material, 3-hydroxypicolinic acid. This application calls for continuing study and extension of the capabilities of 3-hydroxypicolinic acid and the search for new UV light absorbing matrix compounds and mixtures thereof. Experiments also will be performed where the matrix is essentially transparent and the substrate acts as the chromophore for laser desorption. This requires a thin, uniform, reproducible, matrix-analyte film and higher laser powers than used for UV matrices. Optimization of mass resolution will be a major effort of study. Sample preparation and handling, limits of acceptable sample purity, and minimization of sample size are to be studied for optimization as well. Due to the recent developments from this grant, in addition two immediate applications to large-scale sequencing efforts will be investigated early in the grant continuation period. Both require sequencing of oligomers only ~20 to 30 bases long. First, feasibility studies will be performed to assess the potential of mass spectrometry to be automated for rapid screening of entry points of cloned DNA fragments in an undirected (shotgun) strategy to determine if the DNA segment has previously been sequenced. This immediate application would save substantial time and money in large-scale efforts by eliminating much redundant sequencing. Second, we will assess the potential for the technology to resolve ambiguous regions in current gel-based sequencing, such as ambiguities due to compression artifacts.

本研究的主要目的是发展激光解吸时间- 飞行质谱仪变成了一个实用的，非常高速的，DNA 测序技术作为凝胶电泳的直接替代。 质谱仪将测量母体分子质量， 通过Sanger型酶制备的单链DNA寡聚体的嵌套组 化学. 测序中潜在的数量级增长 速度，并降低每基地的成本，源于固有的快速 在飞行时间质谱仪中进行质量分离。 实验安排涉及通过以下方式产生分子离子： 用单个脉冲激光束照射单链DNA， 低聚物混合物分散在主体化学基质中。 的 基质和激光诱导脱附的性质是核心， research. 实现该项目技术目标的主要进展是 特别是我们发现了一种特别有效的 新型紫外线吸收基质材料3-羟基吡啶甲酸 酸 这一应用要求继续研究和推广 3-羟基吡啶甲酸的能力和寻找新的紫外光 吸收基质化合物及其混合物。 实验也将 在基质基本上是透明的并且 底物充当用于激光解吸的发色团。 这需要 薄的、均匀的、可再现的基质分析物膜和更高的激光 比用于UV基质的功率更大。 质量分辨率的优化将 这是一项重大的学习努力。 样品制备和处理，限度 可接受的样品纯度和最小化样品量， 也是为了优化。 由于最近的发展，从这个赠款，另外两个立即 大规模测序工作的应用将在早期进行研究 在补助金持续期间。 两者都需要寡聚体测序 只有约20到30个碱基长。 首先，进行可行性研究 评估质谱分析自动化的潜力， 筛选克隆DNA片段的进入点， （鸟枪）策略，以确定DNA片段是否以前 测序 这种立即应用将节省大量时间， 通过消除大量冗余的排序，在大规模的努力中节省了资金。 其次，我们将评估该技术解决 目前基于凝胶的测序中的模糊区域，例如 由于压缩伪像。