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.

Sequencing of DNA by Gel Electrophoresis in Micromachined Channels

Joe Balch[1], Courtney Davidson[1], Jeff Gingrich[1], Muhammad Sharaf[2], Larry Brewer[1], Jackson Koo[1], Doug Smith[2], Michael Albin[2], and Anthony Carrano[1]
[1]Human Genome Center, L-452, Biology and Biotechnology Research Program, Lawrence Livermore National Laboratory, Livermore, California. 94550, [2]Perkin Elmer Corporation, Applied Biosystems Division, Foster City, California 94404.

Sequencing of DNA by gel electrophoresis is typically performed in slab gel systems. Efforts to increase sequencing rates have generally relied on increasing sample load capacity (higher lane density or multiple capillary systems) and increasing the electric field to obtain faster fragment separation (requiring the use of thinner gels or capillaries to reduce heat dissipation). As an alternative to slab and capillary systems, we are investigating a hybrid technique based upon a high density array of electrophoresis channels fabricated using micromachining technologies on a single, large substrate at fixed locations. Furthermore, standard polyacrylamide gel (PAG) compositions can be poured into the channels using conventional techniques without the problem of bubble formation and other defects commonly incurred in PAG filled capillaries.

We have found that electrophoretic resolution is dependent upon the surface finish of the micromachined channels. We have developed and refined fabrication techniques that result in electrophoretic resolution in microchannels comparable to the electrophoresis resolution measured in standard slab gels formed between two flat glass plates. We have obtained resolution that results in accurate base calling to greater than 500 DNA bases per channel (200 micrometer deep by 1 mm wide by 25 cm long microchannels filled with 6% PAG). Present efforts are underway to develop large electrophoresis channel arrays on a single glass substrate for high throughput DNA sequencing.

This work was performed under a Cooperative Research and Development Agreement (CRADA) between Perkin-Elmer Corporation, Applied Biosystems Division and by Lawrence Livermore National Laboratory under the auspices of the U.S. Department of Energy contract no. W-7405-Eng-48.