GENOME SEQUENCING VIA ION TRAP/TIME-OF-FLIGHT MASS SPEC

通过离子阱/飞行时间质谱进行基因组测序

• 批准号: 2208965
• 负责人: David M. Lubman
• 金额: $ 17.78万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-09-01 至 1998-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2208965
• 关键词: DNA; biomedical equipment development; electrospray ionization mass spectrometry; gas chromatography mass spectrometry; mass spectrometry; nucleic acid sequence

DESCRIPTION (Adapted from Investigator's Abstract): Dr. Lubman proposes to develop new methodology for the rapid sequencing of DNA, involving a combination of biochemical methods and mass analysis via the ion trap storage/reflection time-of-flight hybrid mass spectrometer. The method 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 kbases can be sequenced per day with a desired ultimate goal of 200 kbases 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 throughput. The MALDI process will involve using 3- hydroxypicolinic acid on a Nafion polymer substrate for vaporization and ionization of intact DNA strands. 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 has been interfaced to a reflectron time-of-flight device to allow integration of the signal over multiple laser pulses. In addition, the ion trap storage method has been shown to be essential for attaining enhanced resolution and for minimizing metastable decomposition for the highly energetic ions produced by the MALDI process. Further development of the ion trap/reTOF for storage and analysis of the high mass range required for DNA will be proposed as part of this work. This will involve the use of low frequency RF on the ring electrode and various modifications to the trap and reTOF designs. Ultimately, additional methods for volatilization and ionization of DNA using MALDI from liquid or solid doped matrices will be studied and optimized for these experiments in order to enhance the analytical throughput.

描述（根据调查员的摘要改编）：Lubman博士提出了 开发用于快速测序DNA的新方法，涉及 通过离子陷阱的生化方法和质量分析的组合 存储/反射飞行时间混合质谱仪。该方法 将涉及使用质量分析来测序产生的DNA片段 通过Sanger方法而不是凝胶电泳技术。这 序列编码在产生的DNA链的质量中，可以 根据静电加速迅速分离和鉴定 在飞行器设备中。使用这种方法，据估计 每天至少可以测序50个KBase，并以期望的最终目标进行测序 每天200个KBase。该测序速率将基于矩阵 - 至少的DNA链的辅助激光解吸/电离（MALDI） 200个可以解决和识别的基本对，并且 将提供快速样品吞吐量的多样本解吸探针。 MALDI过程将涉及在Nafion上使用3-羟基甲酸 聚合物底物用于完整DNA链的蒸发和电离。 为了增强该方法检测低的灵敏度 从生化可用 降解，离子陷阱存储技术已连接到 反射率飞行时间设备允许信号整合 多个激光脉冲。另外，离子陷阱存储方法已经 证明对于获得增强的分辨率和 最小化高能离子的亚稳态分解 由Maldi过程产生。离子的进一步发展 陷阱/重新进行存储和分析的高质量范围所需的高质量范围 DNA将作为这项工作的一部分提出。这将涉及使用 环电极上的低频RF和对 陷阱和重新设计。最终，其他方法 使用液体或固体的MALDI对DNA的挥发和电离 掺杂的矩阵将在这些实验中进行研究和优化 为了增强分析吞吐量。