We propose to develop a rapid method for sequencing single-stranded DNA oligomers and polypeptides by reflecting time-of-flight mass spectrometry, using laser desorption, laser ionization, and laser controlled fragmentation. The desorption of DNA segments that have been deposited in a frozen solution or on a solid substrate will be studied by pulsed infrared (IR) and ultraviolet (UV) laser desorption. Desorbed molecules that are neutral in charge will be ionized above the surface by a vacuum ultraviolet (VUV) laser beam, resulting in single-photon ionization; an alternative approach to be compared will be the use of UV laser light, resulting in multiple-photon ionization. Variable fragmentation with an additional laser beam will be investigated as a function of this laser's wavelength, power density, and temporal relationship to the ionizing laser for greatest control in obtaining simple and reliable fragmentation patterns for sequence assignment. The influence of the desorption laser parameters on fragmentation also will be examined.The experiments are designed to decouple as much as possible the three processes of desorption, ionization, and fragmentation for maximum control of these processes. This technology may be capable of sequencing >10(6) nucleotides per day per machine.

Agency
National Institute of Health (NIH)
Institute
National Human Genome Research Institute (NHGRI)
Type
Research Project (R01)
Project #
5R01HG000174-03
Application #
3333221
Study Section
Special Emphasis Panel (SSS (S))
Project Start
1990-08-01
Project End
1993-07-31
Budget Start
1992-08-01
Budget End
1993-07-31
Support Year
3
Fiscal Year
1992
Total Cost
Indirect Cost
Name
Sri International
Department
Type
DUNS #
City
Menlo Park
State
CA
Country
United States
Zip Code
94025
Li, J; Butler, J M; Tan, Y et al. (1999) Single nucleotide polymorphism determination using primer extension and time-of-flight mass spectrometry. Electrophoresis 20:1258-65
Lin, H; Hunter, J M; Becker, C H (1999) Laser desorption of DNA oligomers larger than one kilobase from cooled 4-nitrophenol. Rapid Commun Mass Spectrom 13:2335-40
Monforte, J A; Becker, C H (1997) High-throughput DNA analysis by time-of-flight mass spectrometry. Nat Med 3:360-2
Hunter, J M; Lin, H; Becker, C H (1997) Cryogenic frozen solution matrixes for analysis of DNA by time-of-flight mass spectrometry. Anal Chem 69:3608-12