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The electronic form of this document may be cited in the following style:
Human Genome Program, U.S. Department of Energy, DOE Human Genome Program Contractor-Grantee Workshop IV, 1994.

Abstracts scanned from text submitted for November 1994 DOE Human Genome Program Contractor-Grantee Workshop. Inaccuracies have not been corrected.

Laser Desorption Mass Spectrometry for Fast DNA Sequencing

C. H. Winston Chen,[1] Kai Tang, Nelli I. Taranenko, and Steve L. Allman
Photophysics Group, Chemical Physics Section, Health Sciences Research Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831. [1]Corresponding author.

During the past year, we have achieved some major breakthroughs for using mass spectrometry for DNA analysis. They are (1) successful detection of single-stranded DNA the size of 500 nucleotides and double-stranded DNA the size of 500 base pairs; (2) the detection sensitivity of large DNA segments reaches to the femtomole region, and (3) the first demonstration of using laser desorption mass spectrometry for cystic fibrosis diagnostics. We believe these achievements should become important milestones toward the use of mass spectrometry for fast DNA sequencing. There is a good possibility that fast mass spectrometric DNA sequencing can be demonstrated in the very near future.

Laser desorption mass spectrometry has been considered as a potential new method for fast DNA sequencing. Our approach is to use matrix-assisted laser desorption to produce parent ions of DNA segments and a time-of-flight mass spectrometer to identify the sizes of DNA segments. Thus, the approach is similar to gel electrophoresis sequencing using Sanger's enzymatic method. However, no gel, no radioactive tagging, and no dye labeling are required. In addition, the sequencing process can possibly be finished within a few hundred microseconds instead of hours and days. In order to use mass spectrometry for fast DNA sequencing, the following three criteria need to be satisfied. They are (1) detection of large DNA segments, (2) sensitivity reaches the femtomole region and (3) mass resolution good enough to separate DNA segments of a single nucleotide difference. It has been very difficult to detect large DNA segments by mass spectrometry before due to the fragile chemical properties of DNA and low detection sensitivity of DNA ions. We discovered several new matrices to increase the production of DNA ions. By innovative design of a mass spectrometer, we can increase the ion energy up to 75 KeV to enhance the detection sensitivity. At present, we have fulfilled two key criteria for using mass spectrometry for fast DNA sequencing. The major effort in the near future is to improve the resolution. Different approaches are being pursued. When high resolution of mass spectrometry can be achieved and automation of sample preparation is developed, the sequencing speed to reach 500 megabases per year can certainly be feasible.

In addition to the three major achievements described above, other important accomplished works are:
(1) Success of detecting DNA samples in PCR solutions and Sanger's solution without the need of purification.
(2) Achieve quantitative measurements of DNA by matrix-assisted laser desorption method.
(3) Discovery of the existence of metastable DNA ions.
(4) Detection of restriction enzyme digested DNA.
(5) Detection of point mutation in P-53 gene.
(6) Successful detection of 30 blind samples from cystic fibrosis patients.

Details will be presented in the meeting.

Research sponsored by the Office of Health and Environmental Research, U. S. Department of Energy under contract DEAC05-84OR21400 with Martin Marietta Energy Systems, Inc.