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.

Fluorescent Sequencer Development

William F. Kolbe and Jocelyn C. Schultz
Human Genome Center and Engineering Division, Lawrence Berkeley Laboratory, University of California, Berkeley, CA 94720

In order to address the need for increased DNA sequencing capability, we have initiated a program of gel-based fluorescent sequencer development. Experiments are being performed on two separate platforms. A conventional sized (30 cm x 25 cm x 350 mm) gel apparatus is used to develop improved fluorescence detection methods in order to obtain both high spatial resolution and fast response. This will permit a significant increase in the number of samples which can be loaded on a given gel. At the same time, an ultra-thin gel apparatus (15 cm x 25 cm x 50-100 mm) is being constructed to provide high-speed electrophoretic separations. Detection methods developed in the conventional sized gel apparatus will be incorporated in the ultra-thin gel apparatus as they become available.

The detection system uses a laser beam passing transversely through the gel to excite all of the DNA lanes simultaneously and a fiber optic array to collect the fluorescence produced. A lens array is employed to image the fluorescence onto the ends of the fibers. The output end of the fiber array is formed into a compact rectangular shape compatible with a cooled charge-coupled device camera used to detect the light. The alignment of the laser beam passing through the gel is stabilized by means of a feedback system employing a motorized steering mirror and photodiode detectors positioned at each end of the fiberoptic array. The spatial resolution of the system is sufficient to permit the detection of 100 lanes in a gel width of 25 cm. The system is currently operational with single color detection (one DNA sequence ladder per lane) and is being extended to a four color configuration.

The ultra-thin gel apparatus has been constructed and is undergoing preliminary testing. It should be possible to incorporate the detection system described above in this apparatus in the near future. This combination should lead to greatly enhanced sequencing capabilities.