Optimization of electric field strength for DNA sequencing in capillary gel electrophoresis.
Capillary gel electrophoresis (CGE) has demonstrated the ability to separate DNA sequencing reactions at speeds up to 25 times as great as conventional slab gel electrophoresis. These increased speeds are made possible by the efficient heat dissipation of capillaries, which permits higher electric fields to be employed without deleterious thermal effects. The high electric fields, however, also lead to a reduction in the spacing between bands with a concomitant loss of resolution. The resulting tradeoff between speed and resolution is a very important practical aspect of these high-field separations. This work addresses this question by investigating the band broadening and resolution of DNA fragments as they are separated through a fixed distance of gel at field strengths ranging from 50 to 400 V/cm. It is found that the bandwidths of DNA fragments do decrease with the higher field strengths due to a reduction in the diffusional broadening of bands. However, at sufficiently high electric field strengths, the bands begin to broaden again due to the thermal gradient across the gel. This behavior causes the optimum electric field strength for maximum fragment resolution to depend upon the length of fragments being separated. The relative contributions of diffusion and thermal gradients are discussed and used to predict the ultimate performance of constant field capillary gel electrophoresis.
Luckey
J A
JA
Department of Chemistry, University of Wisconsin, Madison 53706.
Smith
L M
LM
D004058:Diffusion; D004563:Electrochemistry; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D017422:Sequence Analysis, DNA; D013696:Temperature