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.

High-Speed Automated DNA Sequencer Utilizing From-the-Side Laser Excitation

Michael S. Westphall, Robert L. Brumley Jr., Eric C. Buxton, and Lloyd M. Smith
Department of Chemistry, University of Wisconsin, Madison, WI 53706

The Human Genome Initiative is an ambitious international effort to map and sequence the three billion bases of DNA encoded in the human genome. If successfully completed, the resultant sequence database will be a tool of unparalleled power for biomedical research. One of the major challenges of this project is in the area of DNA sequencing technology. At this time, virtually all DNA sequencing is based upon the separation of DNA fragments in high resolution polyacrylamide gels. This method, as generally practiced, is one to two orders of magnitude too slow and expensive for the successful completion of the Human Genome project. One reasonable approach to improved sequencing of DNA fragments is to increase the performance of such gel-based sequencing methods.

Decreased sequencing times may be obtained by increasing the magnitude of the electric field employed. This is not possible with conventional sequencing, due to the fact that the additional heat associated with the increased electric field cannot be adequately dissipated. Recent developments in the use of thin gels have addressed this problem. Performing electrophoresis in ultrathin (50 to 100 microns) gels greatly increases the heat transfer efficiency, thus allowing the benefits of larger electric fields to be obtained. An increase in separation speed of about an order of magnitude is readily achieved. Thin gels have successfully been used in capillary [1] and slab formats [2,3].

A detection system has been designed for use with a multiple fluorophore sequencing strategy in horizontal ultra-thin slab gels. The system employs laser through-the-side excitation and a cooled CCD detector; this allows for the parallel detection of up to 24 sets of four fluorescently labeled DNA sequencing reactions during their electrophoretic separation in ultrathin (115µm) denaturing polyacrylamide gels. Four hundred bases of sequence information is obtained from 100ng of M13 template DNA in an hour, corresponding to an overall instrument throughput of over 9600 bases/hr. A detailed description and the operating characteristics of this system are presented.

[1] Lucky, J. A., Drossman, H., Kostichka, A. J., Mead, D. A., D'Cunha, J., Norris, T. B., and Smith, L. M., Nucleic Acids Research, 18, 4417, (1990)
[2] Brumley, R. L., Jr., and Smith L. M., Nucleic Acids Research, 19, 4121 (1991).
[3] Kostichka, A. J., Marchbanks, M., Bmmley, R. L., Drossman, H., and Smith, L. M., Bio/Technology, 10, 78 (1991).