Stepwise capillary electrophoretic separation of DNA fragments using poly(ethylene oxide) solutions in the presence of electroosmotic flow

Abstract

Single-base resolution in the separation of DNA markers V and VI was achieved in the presence of electroosmotic flow (EOF), using poly(ethylene oxide) (PEO) solutions containing ethidium bromide (EtB) under isocratic conditions. Furthermore, a new approach called stepwise capillary electrophoresis (SCE) has been developed for DNA analysis, including stepwise changes in PEO concentration, EtB concentration as well as both PEO and EtB concentrations, wherein the EOF was used to introduce different PEO solutions into the capillary during the separation. DNA fragments smaller than 80 bp were both detected under isocratic conditions using 20 μg/ml EtB, and SCE using 1 and 20 μg/ml EtB, but not under isocratic conditions using 1 μg/ml EtB. Resolution and speed of the DNA separation in SCE were different from those obtained from isocratic means, indicating that DNA underwent different concentrations of PEO and EtB in SCE. For example, DNA fragments with 458 and 504 base pairs (bp) were partially resolved in SCE, but not under isocratic conditions. The results further suggest that it is worth developing gradient techniques for widening the separation range and enhancing resolution in DNA analysis.