Abstract

We propose to develop efficient high-speed automated methods for the analysis of DNA sequence. Such methods are essential to the success of the Human Genome Initiative, in which the sequence of the human genome will be determined and used as a powerful tool for biological and medical research. Two aspects of sequence analysis will be addressed, the procedures in which DNA for sequencing is produced and fragmented, and the methods employed for separation and analysis. In the first area we will use the Polymerase Chain Reaction method in conjunction with support chemistry to produce pure sets of single-stranded DNA fragments for separation and detection. This chemistry will be automated using a combination of temperature ramping, filtration, and pipetting. We will also investigate the potential of phosphorothioate-based sequencing chemistry in automated sequencing. In the second area capillary electrophoretic separation of the DNA fragments will be investigated for its potential to dramatically increase speed and resolution in automated DNA sequencing.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM042366-01
Application #
3300850
Study Section
(SRC)
Project Start
1989-07-01
Project End
1994-06-30
Budget Start
1989-07-01
Budget End
1990-06-30
Support Year
1
Fiscal Year
1989
Total Cost
Indirect Cost

  Institution

Name
University of Wisconsin Madison
Department
Type
Schools of Arts and Sciences
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715

  Publications

Luckey, J A; Smith, L M (1993) A model for the mobility of single-stranded DNA in capillary gel electrophoresis. Electrophoresis 14:492-501
Kostichka, A J; Marchbanks, M L; Brumley Jr, R L et al. (1992) High speed automated DNA sequencing in ultrathin slab gels. Biotechnology (N Y) 10:78-81
Tong, X; Smith, L M (1992) Solid-phase method for the purification of DNA sequencing reactions. Anal Chem 64:2672-7
Mead, D A; McClary, J A; Luckey, J A et al. (1991) Bst DNA polymerase permits rapid sequence analysis from nanogram amounts of template. Biotechniques 11:76-8, 80, 82-7
D'Cunha, J; Berson, B J; Brumley Jr, R L et al. (1990) An automated instrument for the performance of enzymatic DNA sequencing reactions. Biotechniques 9:80-5, 88-90
Luckey, J A; Drossman, H; Kostichka, A J et al. (1990) High speed DNA sequencing by capillary electrophoresis. Nucleic Acids Res 18:4417-21
Drossman, H; Luckey, J A; Kostichka, A J et al. (1990) High-speed separations of DNA sequencing reactions by capillary electrophoresis. Anal Chem 62:900-3