The power of gene analysis by DNA hybridization methods is now well known. Applications in basic research and in medical areas are growing at a rapid pace. Hybridization probes can be used to detect complimentary sequences of RNA or DNA in fixed cells in isolated chromosomes, and on nitrocellulose filters. The desirable information might be the location of the hybridization probe. For example, one can look for the location of specific genes in chromosomes, or for the locations of infected cells in a tissue biopsy that contains viral RNA or DNA. On the other hand, the desired information might be the quantitative. For example, one might want to measure the amount of a particular RNA in individual cultured cells or the amount of DNA in a cell extract that has been spotted onto nitrocellulose filter paper. There are 3 main methods to visualize the location or amount of nucleic acid probe hybridized to a sample. The probe can be radioactively labeled and detected by counting or autoradiography. The probe can be labeled with a hapten so that addition of enzyme-linked antibodies leads to generation of a colored precipitate at the site of hybridization. A fluorescence microscope or fluorometer can be used to detect the signal from a fluorescent dye that has been attached directly or indirectly to the probe. We believe that the fluorescence detection method has much potential in terms of sensitivity, quantitative accuracy, speed, reproducability, simplicity and versatility. Therefore, we propose to improve the fluorescence hybridization probe detection technology by combining the exciting new cyanine fluorophores that we have already developed with existing techniques and with new methods for linking these fluorophores to nucleic acids. We propose to optimize the technology with a program of thorough probe evaluation and synthetic improvement. We propose to field test the new probes in collaborative projects and to demonstrate the applications.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS019353-10
Application #
2263585
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Project Start
1982-07-01
Project End
1993-03-31
Budget Start
1991-04-01
Budget End
1993-03-31
Support Year
10
Fiscal Year
1991
Total Cost
Indirect Cost
Name
Carnegie-Mellon University
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
052184116
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
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