Introduction to the Workshop
URLs Provided by Attendees

Abstracts

Mapping

Informatics

Sequencing

Instrumentation

Ethical, Legal, and Social Issues

Infrastructure

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.

NEW DIRECTIONS IN HIGH-SENSITIVITY FLUORESCENCE DETECTION OF DNA AND CAPILLARY ARRAY ELECTROPHORESIS

Richard A. Mathies, Jingyue Ju[1], Huiping Zhu, Steven M. Clark, Adarn T. Woolley, Yiwen Wang, Scott C. Benson and Alexander N. Glazer
Chemistry Department and Department of Cell and Molecular Biology, University of California, Berkeley CA 94720.

Capillary Array Electrophoresis, coupled with confocal fluorescence detection,[2] is a valuable new technique for performing high-speed, high-throughput DNA sequencing[3,4] and fragment sizing.[5] In current implementations, up to 50 capillaries can be run in parallel and separations are complete ~10 times faster than slab gels. Our recent efforts have focused on (i) the development of improved fluorescence reagents for DNA sequencing, (ii) the development of methods for using intercalating dyes for double-stranded DNA fragment detection in CE, (iii) and the development of methods for microfabricating capillary arrays.

To improve the spectroscopic properties of the labels used in DNA sequencing, we have synthesized sequencing primers labeled with pairs of dyes that are coupled by fluorescence energy transfer (ET).[6] The donor is chosen to provide intense absorption at the laser wavelength (488 nm) while the acceptors are chosen to provide large Stokes shifts and distinctive emission spectra. The spacing of the dyes along the primer is selected to provide efficient energy transfer with no quenching. The mobility shifts of the ET primers are less than those observed using current commercial dye-labeled primers and the fluorescence intensities are as much as 5 times stronger. Results using these ET dye-labeled primers for 4-color DNA sequencing (collaboration with Dr. Carl Fuller at U. S. Biochemical) and for 2-color allelic fragment detection will be presented.

A variety of monomeric and dimeric intercalating dyes have been used to improve the sensitivity and versatility of DNA fragment detection in CE.[7] The monomeric intercalating dyes TO, TO6, YO, Propidium 2 and Propidium 3 have been used for on-column staining. TO and TO6 are able to detect as little as 1 fg/µL of a 600 bp fragment in the initial sample. Separations of DNA precomplexed with the dimeric intercalating dyes EthD, TOTAB, and YOYO have also been successful, although it is important to work at low dye:DNA ratios and to use 9-aminoacridine in the running buffer to achieve high resolution separations.

In a continuing effort to miniaturize DNA analysis systems, we have used photolithographic techniques to microfabricate capillary arrays.[8] These arrays were fabricated on planar glass slides by first etching channel patterns, and then forming the capillaries by thermally bonding the etched substrate to a top glass plate. The channels have an effective length of 3.5 cm and the 10 micron deep channels ranged from 30-120 µm in width. Using these CAE chips, high-resolution separations of double-stranded (PHI)X/HaeIII DNA have been performed from 70-1000 bp in only 120 seconds! Since up to 100 such channels can be fabricated on a single glass slide, this work establishes the feasibility of developing miniaturized DNA analysis chips.

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-FG-91-61125.

[1]DOE Human Genome Distinguished Postdoctoral Fellow
[2]R.A. Mathies et al., Rev. Sci. Instrum. 65, 807-812 (1994).
[3]R.A. Mathies and X.C. Huang, Nature (London) 359, 167-168 (1992).
[4]R.A. Mathies and X.C. Huang, Automated DNA Sequencing and Analysis, eds. M.D. Adams, C. Fields & J. C. Venter, Academic Press, pp. 17-28 (1994).
[5]S.M. Clark and R.A. Mathies, Anal. Biochem. 215, 163-170 (1993).
[6]J. Ju, C. Fuller, C. Ruan, A.N. Glazer and R.A. Mathies, Fluorescence Energy Transfer Primers for DNA Sequence Analysis, in preparation.
[7]H. Zhu, S.M. Clark, S.C. Benson, H.S. Rye, A.N. Glazer and R.A. Mathies, Analytical Chemistry 66, 1941-1948 (1994).
[8]A.T. Woolley and R.A. Mathies, Ultra-High-Speed DNA Fragment Separations Using Microfabricated Capillary Array Electrophoresis Chips, Proc. Natl. Acad. Sci. U.S.A., in press.