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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.
DESIGN, SYNTHESIS AND CHARACTERIZATION OF ENERGY TRANSFER FLUORESCENT DYE TAGGED OLIGONUCLEOTIDE PRIMERS FOR DNA SEQUENCE ANALYSIS
Jingyue Ju, Chihchuan Ruan, Carl W. Fuller, Alexander N. Glazer and Richard A. Mathies
Department of Chemistry and Department of Cell and Molecular Biology, University of California, Berkeley, CA 94720-1460. United States Biochemical Corporation, P.O. Box 22400, Cleveland, Ohio 44122
The objective of our work is to develop novel fluorescently labeled primers for DNA sequencing and other types of multicolor genetic analysis that allow higher levels of detection sensitivity. We describe here: (1) the design and synthesis of energy transfer (ET) fluorescent dye-labeled oligonucleotide primers with improved spectroscopic and electrophoretic properties; (2) The evaluation of these primers in DNA sequencing on an ABI 373A DNA sequencer and a capillary array electrophoresis (CAE) system. Specifically, energy transfer fluorescent dyes (donor and acceptor) are incorporated on the M13 (- 40) universal primer. The primers are denoted as D-N-A, where D is the donor, A is the acceptor, and N is the number of bases between D and A. In all the primers prepared, the donor is the fluorescein derivative FAM (F) incorporated at the 5' end of the oligonucleotide. The acceptors are the commercially available dyes JOE (J), TAMRA (T) or ROX (R) incorporated at the location of a modified T in the primer sequence. Twenty ET primers with the same donor chromophore and different acceptors having different donor-acceptor separations were synthesized and purified. The evaluation of the spectroscopic and electrophoretic properties of these primers led to an elegant way of tuning the mobility shift of the primers on electrophoresis as well as achieving optimum fluorescence intensities. This is achieved by adjusting the spacing of the donor and acceptors to get a similar mobility match and still maintain good energy transfer. Four of the primers (F-10-F, F-10-J, F-3-T and F-3-R) with optimal properties were selected for four-color DNA sequencing on an ABI 373A sequencer. The fluorescence intensity of the DNA sequencing fragments generated with ET primers is 2-5 times higher than that of the corresponding fragments generated with the conventional single dye-labeled primers. In a typical sequencing run, a 500-base sequence is determined with an accuracy of >99%. The design, synthesis, purification and spectroscopic properties of ET primers, the way of adjusting the electrophoretic properties of the ET primers, and the DNA sequencing protocol with ET primers will be presented.
This research was supported by a grant from the Director, Office of Energy Research, Office of Health and Environmental Research of the U.S. Department of Energy under contract DE-FG91-61125. J. J. was supported by a Human Genome Distinguished Postdoctoral Fellowship sponsored by the U.S. Department of Energy, Office of Health and Environmental Research, and administered by the Oak Ridge Institute for Science and Education. Support from United States Biochemical Corporation is also gratefully acknowledged.
J. Ju, C. W. Fuller, C. Ruan, A. N. Glazer and R. A. Mathies, Fluorescence Energy Transfer Primers for DNA Sequence Analysis, in preparation.