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.

Toward an Automated System for High-Throughput DNA Sequencing: 2. Multiple Capillary Electrophoresis with a Replaceable Matrix

Mark A. Quesada, Jan Kieleczawa, Shiping Zhang, John J. Dunn and F. William Studier
Biology Department, Brookhaven National Laboratory, Upton, New York 11973

Primer walking on multiple templates, using primers supplied from a hexamer library, has the potential for sustained, high-throughput production of sequencing reactions [1]. A capillary electrophoresis and fluorescence detection system is being developed to provide the capacity and sensitivity needed to take advantage of this potential. Our initial goal is to read several hundred bases in less than an hour from sequencing reactions primed by hexamer strings, using the ABI four-color fluorescent terminators and a replaceable resolving matrix of linear polyacrylamide [2]. Ultimately, we plan to analyze many capillaries in parallel, using complete fluorescence spectra to determine the base sequence and assign confidence levels.

A prototype single-capillary electrophoresis system with four-color detection was constructed and used to assess requirements for analyzing the products of sequencing reactions primed by hexamer strings. We are collaborating with Karger's group at Northeastern University to implement capillary electrophoresis with replaceable linear polyacrylamide matrices. Results will be presented detecting the products of sequencing reactions primed by hexamer strings and terminated by ABI four-color fluorescent terminators, priming at different sites in M13 templates.

Preliminary work toward developing a multiple capillary system will also be presented. Fiber-optic illumination and detection is being tested relative to a confocal system. Multiple fiber-optic output to a spectrograph would allow full spectral detection of many capillaries in parallel on a CCD, collecting data fast enough to read 400 or more bases an hour from each capillary. A potential configuration for a fully automated multiple capillary system will be described.

[1] Kieleczawa, J., Dunn, J. J., and Studier, F. W. (1992) DNA sequencing by primer walking with strings of contiguous hexamers. Science, 258, 1787-1791.
[2] Ruiz-Martinez, M. C., Berka, J., Belenkii, A., Foret, F., Miller, A. W., and Karger, B. L. (1993) DNA sequencing by capillary electrophoresis with replaceable linear polyacrylamide and laser-induced fluorescence detection. Anal. Chem. 65, 2851-2858.