W.H. Benner and J.M. Jaklevic
Ernest Orlando Lawrence Berkeley National Laboratory, University of California, Engineering Science Department, Human Genome Group, 1 Cyclotron Road, Berkeley, CA 94720, Phone: (510) 486-7194, Fax: (510) 486-5857, whbenner@lbl.gov
Mass spectrometry is an instrumental method capable of producing rapid analyses with high mass accuracy. When applied to genome research, it is an attractive alternative to gel electrophoresis. At present, routine DNA analysis by mass spectrometry is seriously constrained to small DNA fragments. Contrasted to other mass spectrometry facilities in which the development of ladder sequencing is emphasized, we are exploring the application of mass spectrometry to procedures that identify short sequences. This approach helps the molecular biologists associated with LBL's Human Genome Center to identify redundant sequences and vector contamination in clones rapidly, thereby improving sequencing efficiency. Biological presequencing procedures designed to use mass spectrometry as a detection scheme will be presented as ways to perform oligonucleotide ligation assays, end-of-fragment sequencing and the sizing of deletion series created in P1, BAC and PAC vectors.
We are also working to improve the operation of matrix-assisted-laser-desorption-ionization (MALDI) and electrospray mass spectrometers by developing new ion detectors. One of the limitations for applying mass spectrometry to DNA analysis \relates to the poor efficiency with which conventional electron multipliers detect large ions, a problem most apparent in MALDI-TOF-MS. To solve this problem, we are developing alternative detection schemes which rely on heat pulse detection. The kinetic energy of impacting ions is converted into heat when ions strike a detector and we are attempting to measure indirectly such heat pulses. We are developing two detectors based on impact detection. We have generated particle impact signals in metal-oxide-silicon structures and a piezoelectric film.
Electrospray ion sources generate ions of megadalton DNA with minimal fragmentation, but the mass spectrometric analyses of these large ions usually leads only to a mass-to-charge distribution. If ion charge were known, actual mass data could be determined. To address this problem, we are developing a detector that will simultaneously measure the charge and velocity of individual ions. Mass spectra of megadalton DNA will be presented showing the feasibility for sizing fragments in the 2 to 40 kb region.
This work was supported by the Director, Office of Energy Research, Office of Health and Environmental Research, Human Genome Program, of the U.S. Department of Energy under Contract No. DE-AC03-76SF00098.