Effect of glycerol-induced DNA conformational change on the separation of DNA fragments by capillary electrophoresis

The effect on DNA separation of adding glycerol to the running buffer was studied using linear polyacrylamide (LPA) or poly(ethylene oxide) (PEO) as separation medium. For both LPA and PEO, it was found that the addition of 25% (v/v) glycerol to the running buffer enhanced the separation of large double-stranded DNA fragments and increased the migration time. The two buffers used, 1×TBE (Tris–boric acid–EDTA) and 1×TTE (Tris–N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS)–EDTA), showed similar improvement, but the effect on the 1×TBE buffer was more amplified. The difference in buffer properties, such as viscosity, conductance, and pH, had little effect on the separation. We attribute the improvements made in the separation to the ability of glycerol to induce a conformational change in DNA as demonstrated by dynamic light scattering results. The presence of glycerol can increase the electrostatic interactions between the phosphate groups, decrease the hydration sphere of the polynucleotides, and compete with water to form hydrogen bonds with the side group of bases. These interactions increase the DNA contour length and reduce the effective charge over weight ratio, which can explain the experimental data. The complex formed by boric acid and glycerol had a stronger effect on the DNA conformation change than glycerol itself. This enhancement was also observed in DNA sequencing analysis.