Gel electrophoresis of DNA fragments in narrow-bore capillaries

In this work, we studied the behavior of electrophoretic columns, having an inner diameter (ID) of 2-10 mu m, filled with a cross-linked polyacrylamide gel matrix. The usefulness of these columns for DNA sequencing is discussed. Evaluation of column performance included tests of gel stability and migration time reproducibility. Confocal laser-induced fluorescence (LIF)-detection was employed utilizing a 488 nm argon ion laser for separations of C- and T-terminated DNA Sanger fragments. Reducing the inner diameter of the column from 50 mu m to 10 mu m resulted in an approximately eightfold increase in lifetime, under conditions in which the columns were subjected to a field strength of 1000 V/cm. The 10 mu m ID columns were utilized for separation of Sanger fragments, and adequate detection sensitivity was obtained by stacking of the fragments from a deionized sample solution. A linear algorithm for retention data synchronization between individual electropherograms was employed to provide a route towards a reliable automated base calling protocol.