SEQUENCING VIA ION-TRAP/TIME-OF-FLIGHT MASS SPECTROMETRY

我们建议开发新的DNA快速测序方法， 涉及生化方法和质谱学的结合 分析。这将涉及到使用质量分析来对DNA进行排序 用桑格法而不是凝胶电泳法产生的片段 技巧。该序列在大量的DNA链中被编码 产生的，可以根据以下条件快速分离和鉴定 飞行时间装置中的静电加速。使用这个 方法学估计至少有50个K碱基可以被测序 一天，期望的最终目标是每天200个K碱基。这个测序 费率将基于基质辅助激光解吸/电离 (MALDI)至少200个碱基对的DNA链，可以是质量的 以及一个多样品解吸探头 全程提供快速样品。为了提高其敏感性， 检测每条DNA链低分子水平的方法 可用于生化降解，离子陷阱存储技术将 与反射飞行时间装置相连接。此外， 离子陷阱存储方法将是获得足够的 MALDI过程产生的高能离子的分辨率。 最终，这项工作中要探索的一个关键问题将涉及 DNA挥发和电离方法的研究进展 来自液体或固体掺杂基质的MALDI，将被研究和 为这些实验进行了优化。

We propose to develop new methodology for the rapid sequencing of DNA, involving a combination of biochemical methods and mass spectroscopic analysis. This will involve using mass analysis to sequence the DNA fragments produced by the Sanger method rather than gel electrophoretic techniques. The sequence is encoded in the mass of the DNA strands produced, which can be rapidly separated and identified based upon electrostatic acceleration in a time-of-flight device. Using this methodology it is estimated that at least 50 K bases can be sequenced per day with a desired ultimate goal of 200 K bases per day. This sequencing rate will be based upon matrix-assisted laser desorption/ionization (MALDI) of DNA strands of at least 200 base pairs which can be mass resolved and identified and a multisample desorption probe which will provide rapid sample throughout. In order to enhance the sensitivity of the method to detect the low femtomolar levels of each DNA strand available from biochemical degradation, ion trap storage technology will be interfaced to a reflection time-of-flight device. In addition, the ion trap storage method will be essential for attaining sufficient resolution for the highly energetic ions produced by the MALDI process. Ultimately, a key issue to be explored in this work will involve the development of methods for volatilization and ionization of DNA using MALDI from liquid or solid doped matrices which will be studied and optimized for these experiments.