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

我们建议开发新的DNA快速测序方法， 包括结合生物化学方法和质谱 分析. 这将涉及使用质量分析来测序DNA 用桑格法而不是凝胶电泳法产生的片段 技术. 序列被编码在大量的DNA链中 产生的，可以快速分离和识别的基础上， 飞行时间装置中的静电加速。 使用此 方法学，估计至少50 K碱基可以测序，每个 最终目标是每天20万个碱基。 该测序 速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，其可以是质量 解析和识别，以及多样品解吸探针， 提供全程快速采样。 为了提高灵敏度， 检测每条DNA链的低飞摩尔水平的方法 可从生化降解，离子阱存储技术将 连接到反射飞行时间装置上 此外该 离子阱存储方法对于获得足够 MALDI过程产生的高能离子的分辨率。 最终，这项工作中要探讨的一个关键问题将涉及 开发了使用DNA的挥发和电离方法 将研究来自液体或固体掺杂基质的MALDI， 优化这些实验。

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.