Luckey J A, Smith L M
Department of Chemistry, University of Wisconsin, Madison 53706.
Anal Chem. 1993 Oct 15;65(20):2841-50. doi: 10.1021/ac00068a022.
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.
毛细管凝胶电泳（CGE）已证明能够以比传统平板凝胶电泳快25倍的速度分离DNA测序反应。毛细管高效的散热能力使得这些更高的速度成为可能，这允许使用更高的电场而不会产生有害的热效应。然而，高电场也会导致条带间距减小，同时分辨率降低。速度和分辨率之间的这种权衡是这些高场分离非常重要的实际方面。这项工作通过研究DNA片段在50至400 V/cm场强下通过固定距离凝胶分离时的条带展宽和分辨率来解决这个问题。研究发现，由于条带扩散展宽的减少，DNA片段的带宽确实会随着场强的增加而减小。然而，在足够高的电场强度下，由于凝胶上的热梯度，条带又开始展宽。这种行为导致实现最大片段分辨率的最佳电场强度取决于被分离片段的长度。文中讨论了扩散和热梯度的相对贡献，并用于预测恒场毛细管凝胶电泳的最终性能。
