SEQUENCING OF DNA BY LASER IONIZATION

通过激光电离进行 DNA 测序

• 批准号: 3333219
• 负责人: CHRISTOPHER H BECKER
• 金额: $ 31.96万
• 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/3333219
• 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.

这项研究的主要目的是开发激光解吸时间-时间 飞行质谱学进入一个实用的，非常高速的DNA 测序技术作为凝胶电泳的直接替代品。 质谱仪将测量母体分子质量 Sanger-type制备的嵌套式单链DNA低聚物 化学反应。测序中潜在的数量级增长 速度和每个基地成本的降低源于固有的快速 在飞行时间质谱仪中进行的质量分离。 实验安排包括通过以下方式产生分子离子 单束脉冲激光照射单链DNA 分散在主体化学基质中的低聚物混合物。这个 基质的性质和激光诱导的解吸是 研究。该项目技术目标的主要进展是 尤其是我们发现了一种特别有效的 新型紫外光吸收基质材料3-羟基烟酸 酸。 这项申请需要继续研究和延长 3-羟基烟酸的性能及寻找新的紫外光 吸收基质化合物及其混合物。实验也将 在矩阵本质上是透明的，并且 底物是激光解吸的生色团。这需要 一种薄的、均匀的、可重现的基质分析薄膜和更高的激光器 比用于UV矩阵的功率。质量分辨率的优化将 是一项主要的研究工作。样品制备和处理的限值 可接受的样品纯度和最小化的样品大小 并对其进行了优化研究。 由于最近这笔赠款的发展，另外两个立即 大规模测序工作的应用将在早期进行调查 在赠款续行期内。两者都需要对低聚物进行测序 只有20到30个碱基长。首先，将进行可行性研究 评估质谱学在快速检测中实现自动化的潜力 非定向克隆DNA片段切入点的筛选 (猎枪)策略，以确定DNA片段之前是否 已排序。这一即时申请将节省大量时间，并 通过消除大量多余的测序，在大规模工作中投入资金。 其次，我们将评估该技术解决方案的潜力 当前凝胶测序中的歧义区域，如歧义 由于存在压缩伪影。