SEQUENCING OF DNA BY LASER IONIZATION

本研究的主要目的是研究-的激光解吸时间

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.