Danhua Chen, Mark D. Peterson, Robert L. Brumley, Jr., Michael C. Giddings, Eric C. Buxton, Michael S. Westphall, Lloyd Smith* and Lloyd M. Smith*
Department of Chemistry, University of Wisconsin-Madison. Madison WI 53706.
Electrophoresis in thin gels provides increased heat transfer efficiency, permitting larger electric fields to be employed with correspondingly more rapid separations.[l,2] This is of particular interest in the area of fluorescence-based automated DNA sequence analysis, where there is a tremendous need for increased throughput from sequencing instruments.[3] Utilizing a four color cooled CCD camera in conjunction with ultrathin slab gels, Kostichka et al. demonstrated an order-of-magnitude increase in separation speed for fluorescence-based DNA sequencing.[4]
This prototype apparatus, which had a narrow field of view, was optimized to include a larger CCD chip in the detection system. This allowed a factor of three increase in the imaged area, thus allowing additional samples to be run in parallel. Sample excitation for this the system was accomplished by bringing the laser beam into the gel from the side. This method of excitation permitted the required excitation power density to be obtained with an air cooled argon ion laser, as the excitation beam cross section remains small. The amount of background fluorescence is also reduced since the beam does not pass through the glass used to cast the sequencing gel.
This approach has been used successfully for conventional sequencing gels about 400 microns in thickness, and is employed in commercial sequencing instruments from Hitachi and Pharmacia. However, the fundamental properties of gaussian laser beams introduce problems when trying to pass the beam through an ultrathin gel. Namely, the tighter the focus of the beam the shorter the distance over which the focus can be maintained. Fortunately, the high efficiency of grazing incidence reflection effectively traps the beam between the glass plates, resulting in a high throughput of the laser energy.
A theoretical model describing the beam throughput has been developed. In this model attenuation of the beam intensity is attributed to four factors: aperturing at the entrance of the gel; reflective losses upon entrance into the gel; scattering during transmission through the gel; and reflective losses occurring upon successive "bounces" of the beam from the gel-glass interface during propagation of the beam. The beam properties as characterized theoretically are shown to be in good agreement with the experimentally determined values.
*Supported by a grant from the Director, Office of Energy Research, Office of Health and Environmental Research of the U.S. Department of Energy under contract DE-FG02-90ER61026
**Lawrence Berkeley Laboratory, Berkeley CA.
[1] Drossman, H.; Luckey, J. A.; Kostichka, A. J.; D'Cunha, J.; Smith, L. M. Anal. Chem. 1990, 62, 900-903.
[2] Brumley, R. L.; Smith, L. M. Nucl. Acids Res. 1991, 19, 4121-2126.
[3] Hunkapiller, T.; Kaiser, R. J.; Koop, B. F.; Hood, L. Science. 1991,254, 59-67.
[4] Kostichka, A. J.; Marchbanks, M. L.; Brumley, R. L.; Drossman, H.; Smith, L. M. Bio/Technology. 1992, 10, 78-81.