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.

Capillary Array Electrophoresis for High Throughput DNA Analysis

John Bashkin, David Barker, Dave Roach, Matt Bartosiewicz, Joe Leong, Tom Zarella, Rick Johnston
Molecular Dynamics, Sunnyvale, CA 94086

Capillary electrophoresis can be used effectively for rapid DNA analysis. Applications include DNA sequencing, diseases diagnostics, genetic typing, and genome mapping. However, experimental throughput has been limited by the requirement of running samples sequentially down a single capillary. Expanded use of the technique has also been hindered by the cost and reliability of polyacrylamide-filled capillaries, and the poor resolution achieved by currently available replaceable matrices. We have extended the work of R. Mathies [1] and developed a scanning instrument capable of electrophoresis and analyte detection in 48 capillaries simultaneously.

Our capillary array electrophoresis (CAE) instrument detects DNA through Laser Induced Fluorescence (LIF), employing an Ar+ laser at 488nm. Hydroxyethyl cellulose of various molecular weights and percentages are used as the separation matrix, or sieving buffer. The system is connected to either a vacuum or pressure source to introduce the matrix into the capillaries rapidly and conveniently. The total turnaround time for replacement of the sieving buffer between electrophoretic separations is approximately 20 minutes. We present data on separations of dsDNA restriction fragments and PCR products, with single base-pair resolution over 100-400bp, and a total size range of 50bp-12kbp. The fragments are fluorescently labeled by inclusion of thiazol orange in the sieving buffer. Total experimental run time from sample injection to resolution of fragments up to 2kbp is 20-30 minutes. The electrical current is monitored in all capillaries during an experiment. This data is stored and can be used to correct for slight differences in the electrical power delivered to each capillary across the array.

Data analysis software, ArrayQuant(tm), has also been developed under the WindowsNT(tm) environment. This package accomplishes automatic peak detection, fragment sizing, and error analysis for the electropherograms. Data reports for all 48 capillaries are generated semiautomatically and conveniently transferred to Excel(tm) spreadsheets.

This work was funded by a Department of Energy SBIR Phase II Grant (DE-FG03-92 ER81299, D. Barker, P.I.).

[1] Huang, X.C., Quesada, M.A., Mathies, R.A. (1992) DNA Sequencing Using Capillary Array Electrophoresis. Anal. Chem., 64, 2149-2154.