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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.

Three Dimensional Imaging of DNA Fragments During Electrophoresis Using a Confocal Detector

Larry Brewer, Courtney Davidson, Joe Balch, and Anthony Carrano
Human Genome Center, L-452, Biology and Biotechnology Research Program, Lawrence Livermore National Laboratory, Livermore, California. 94550

We have measured the three dimensional distribution of individual fragments of DNA during electrophoresis. Separations (65 V/cm) were carried out in a 6% polyacrylamide gel confined by a glass microchannel with cross sectional dimensions of 200 microns deep by 1000 microns wide. The detection system consisted of a confocal microscope with a measured depth of focus of 30 microns (FWHM) plus a photomultiplier to detect the laser induced fluorescence from the dye-labeled DNA. The photomultiplier detector was used in a photon counting mode because of the small signal levels (~1000 Hz). The electrophoresis microchannel plates were mechanically scanned using a precision X-Y stage and a Z axis microscope focus controller, both under computer control. Preliminary results indicate that the DNA was confined in the center of the microchannel, with an approximately Gaussian profile along all three axes. DNA fragments ranging from 50 to 300 bases were profiled and found to have similar spatial distributions. These results will be important for designing optimized high-throughput electrophoresis systems.

This work was performed under the auspices of the U.S. Department of Energy by Lawrence Livermore National Laboratory contract no. W-7405-Eng-48.