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Capillary gel electrophoresis with fluorescence detection is a new technique for rapid DNA sequence analysis. The state of this technology and the potential for its future use are discussed.
The tremendous demands of the Human Genome Initiative for improved DNA sequencing methodologies has spurred a multitude of innovative new approaches to high-speed DNA sequence analysis. One of the most promising of these new techniques is capillary gel electrophoresis (CGE). This method is a variant of capillary zone electrophoresis (CZE), discussed in an earlier product review1.
As in CZE, electrophoretic separations are performed in extremely thin (1 to 100microns i.d.) fused silica capillaries; the difference is that in CGE the capillary is filled with a polyacrylamide gel matrix to permit separation on the basis of molecular weight, as in conventional gel electrophoretic separations. The principal advantage of the method is its speed - the electrical resistance of these thin capillaries is sufficiently great that electric fields as large as 400 V cm'1 (compared to the 30 or so volts per cm used in conventional DNA sequencing gels) can be applied to them without excessive Joule heating. These large fields result in a corresponding increase in the migration velocity of the DNA fragments, with a concomitant reduction in analysis time2'4 (Fig. 1).
Resolution
The resolutions obtained in these CGE separations are also extremely high, in the range of 10 to 30 million theoretical plates per metre2 4 5. There is, as yet, a lack of consensus on whether these results differ materially from those obtained under similar conditions in conventional slab gel electrophoresis. Data from our group have shown roughly similar resolving power in the two methods4, whereas results reported elsewhere showed a greater difference2. The apparent discrepancy seems to be due primarily to variations in the conditions of the conventional electrophoresis, as both groups obtained similar results using CGE. In any case, band broadening is clearly not diffusive in nature, as turning off the electrophoresis mid-run, even for several hours, has no effect on band widths. The relative importance of other broadening mechanisms, such as random variations in the path lengths taken by DNA molecules through the gel or sample injection effects,...