ANALYSIS OF RESOLUTION IN DNA SEQUENCING BY CAPILLARY GEL-ELECTROPHORESIS

DNA sequence analysis is based upon the electrophoretic separation of DNA fragments by denaturing polyacrylamide gel electrophoresis. It is necessary to understand the factors which determine resolution in these separations in order to optimize performance. Resolution is determined by the width of the DNA bands and the spacing between the bands. In this paper, resolution is studied using capillary gel electrophoresis in a nonstacking buffer system. It is shown that the width of the DNA bands is determined by four factors: injection, diffusion, thermal gradients, and detection volume. The relative contribution of each effect is determined as a function of fragment length. This information is used in conjunction with empirically determined mobilities to predict resolution as a function of capillary length at a fixed electric field. The implications of this analysis for optimum separations are discussed.