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.

MULTIPLE CAPILLARY DNA SEQUENCING

Norman J. Dovichi[1,2,3], Jian-Zhong Zhang[1], Jian-Ying Zhao[1], Jiang Rong[1], Rong Liu[1], John Elliott[2], Sue Bay[1], Pieter Roos[1], and Larry Coulson[1]
[1]Department of Chemistry and [2]Department of Medical Microbiology, University of Alberta, Edmonton, Alberta CANADA T6G 2G2. [3]corresponding author.

We have developed a multiple capillary DNA sequencer. This instrument has several important attributes. First, by operation at an electric field of 200 V/cm, we are able to separate DNA sequencing fragments rapidly and efficiently. Typically, 1.5 hours are required to separate fragments up to 550 bases in length. Second, the separation is performed with 5%T 0%C polyacrylamide. This non-crosslinked matrix has sufficiently low viscosity that it can be pumped from the capillary and replaced with fresh material when required. Third, the fluorescence detection cuvette is manufactured locally by means of microlithography technology. This detection cuvette provides robust and precise alignment of the optical system. Fourth, the system is based on Applied Biosystems' four color sequencing technology. We routinely separate both PRISM-labeled dideoxynucleotide terminated fragments and conventional primer-labeled sequencing fragments. The majority of molecular biologists are familiar and comfortable with this labeling technology, which should enhance the acceptance of the sequencer by the user community. Fifth, we use fiber-optic coupled avalanche photodiode photodetectors, which provide low noise and high sensitivity detection. We have obtained detection limits of 120 fluorescein molecules injected onto the capillaries. We operate a number of capillaries simultaneously, increasing the throughput of the system. The system operates with 5 to 32 capillaries; the five capillary device is ideally suited for small-scale sequencing projects, while the larger instruments are appropriate for genomic scale sequencing. Finally, we have developed an automated DNA base-calling algorithm.

This combination of speed, flexibility, and labeling technology provides the first example of a practical and useful DNA sequencer based on capillary electrophoresis. The instrument is very robust, and has been routinely used for six months. We have begun to sequence a number of templates, and we will report sequencing accuracy and sequence read length at the conference. We are currently using an exonuclease method to synthesize a set of nested deletions in a directed sequencing strategy. This approach greatly simplifies sequence assembly problems compared with a shot-gun sequencing strategy.

A next-generation system is under construction that will operate 96 capillaries simultaneously. This system will eventually be expanded to 864 capillaries. An 864 capillary instrument will produce nearly half a million bases of raw sequence data in a 1.5 hour run. Our automated base-calling algorithm is being developed for the 864-capillary instrument; the algorithm is very fast, requiring less than a second for a typical sequencing run.