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.

Application of Single Molecule Detection to DNA Sequencing and Sizing

W. Patrick Ambrose, Peter M. Goodwin, James H. Jett, Mitchell E. Johnson, John C. Martin, Babetta L. Marrone, Jeffery T. Petty, Jay A. Schecker, Ming Wu, Richard A. Keller
Center for Human Genome Studies, Los Alamos National Laboratory, Los Alamos, NM 87545

Alberto Haces, Po-Jen Shih, and John D. Harding
Corporate Research and Molecular Biology Research and Development, Life Technologies Inc. (GIBCO BRL), 8717 Grovemont Circle, Gaithersburg, MD 20898

We are developing a laser-based technique for the rapid sequencing of 40-kb or larger fragments of DNA at a rate of 100 to 1000 bases per second. Our approach relies on fluorescent labeling of the bases in a single fragment of DNA, attachment of this labeled DNA fragment to a support, movement of the supported DNA into a flowing sample stream, and detection of the individual fluorescently labeled bases by laser-induced fluorescence as they are cleaved from the DNA fragment by an exonuclease. The ability to sequence large fragments of DNA will reduce significantly the amount of subcloning and the number of overlapping sequences required to assemble megabase segments of sequence information. Current status will be presented.

We are also applying our sensitive fluorescence detection to sizing of DNA fragments. Large, fluorescently stained restriction fragments of lambda phage DNA are sized by passing individual fragments through a focused, continuous- wave laser beam in an ultrasensitive flow cytometer at a rate of ~60 fragments per second. The size of the fluorescence burst emitted by each stained fragment as it passes through the laser beam, is measured in one millisecond. We have demonstrated flow cytometric sizing of DNA fragments in a ~0.1-pg sample of a restriction digest of lambda DNA in 164 seconds with sizing accuracy better than 98%. Our current sizing range is 2 kb to 150 kb.