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.

DNA ANALYSIS BY CAPILLARY ELECTROPHORESIS: SEQUENCING AND MUTANT DETECTION

Barry L. Karger, Jan Berka, Marie C. Ruiz-Martinez, Steve Carson, Appasani S.M. Krishnarao, Kirstin Hebenbrock and Frantisek Foret
Barnett Institute, Northeastern University, Boston, MA 02115

A core technology based on capillary electrophoresis with a replaceable linear polyacrylamide sieving matrix and laser-induced fluorescence detection has been developed for DNA analysis. We will first report on the application of this technology to DNA sequencing. Specifically, we are using the modular primer-walking strategy and dye-labelled terminators for sequencing stretches of DNA at a minimum rate of 500 bases/hr. The design of a fully automated multiple capillary instrument which can replace the polymer matrix after each run will be shown. In this design, emphasis has been placed on flexibility, for use with a variety of excitation and emission wavelengths, while maintaining low detection limits and high spectral resolution. The front-end automation for primer selection, Sanger reaction, sample clean-up and injection will also be described.

A second topic will deal with high resolution separation and analysis of point mutations, using the above core technology. Both SSCP and CDCE (constant denaturant capillary electrophoresis) have been developed utilizing multiple dye-labelling strategies. The excellent resolution of heteroduplexes from homoduplexes in CDCE permits detection of mutants in low frequency relative to wild type (< 1 part per thousand). With appropriate post-column collection, it is possible to sequence individual species for mutant structure determination.