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.
