Novel elution strategy for monitoring DNA counter-migration in the presence of electroosmotic flow

Abstract

The migration behavior of native (i.e., unlabelled) DNA in the presence of electroosmotic flow (EOF) was investigated in bare fused-silica capillaries. Employing a novel elution strategy, the influence of EOF on the net mobility of DNA was assessed by collecting the DNA that migrated anodically (i.e., against EOF) and out of the capillary inlet. Various conditions of pH and buffer-zone continuity were employed to characterize this phenomenon. Tris acid (TA, pH 5.14) and Tris base (TB, pH 9.36) were used as buffers in continuous systems, in which the capillary and the inlet reservoir contain the same buffer, and discontinuous systems, in which the capillary contains either TA or TB, and the inlet reservoir contains water. DNA that was ejected into the inlet vial was subsequently analyzed by capillary electrophoresis–laser-induced fluorescence. Both ΦX174/ HaeIII DNA and the β-actin product of single-cell reverse transcriptase-polymerase chain reaction were used as DNA samples in this study. The mechanism of elution was found to depend on bulk flow, in the case of continuous solutions. However, with the discontinuous system, a localized decrease in EOF generated in the capillary tip appeared to impact elution. These findings serve to introduce an alternative approach for characterizing the mobility of highly charged species.