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 s tudied using linear polyacrylamide (LPA) or p 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 buf fers used, 1 x TBE (Tris-boric acid-EDTA) and 1 x TTE (Tris-N-tris(hydroxym ethyl)methyl-3-aminopropanesulfonic acid (TAPS)-EDTA), showed similar impro vement, but the effect on the 1 x TBE buffer was more amplified. The differ ence in buffer properties, such as viscosity, conductance, and pH, had litt le effect on the separation. We attribute the improvements made in the sepa ration to the ability of glycerol to induce a conformational change in DNA as demonstrated by dynamic light scattering results. The presence of glycer ol can increase the electrostatic interactions between the phosphate groups , decrease the hydration sphere of the polynucleotides, and compete with wa ter to form hydrogen bonds with the side group of bases. These interactions increase the DNA contour length and reduce the effective charge over weigh t ratio, which can explain the experimental data. The complex formed by bor ic acid and glycerol had a stronger effect on the DNA conformation change t han glycerol itself. This enhancement was also observed in DNA sequencing a nalysis. (C) 2001 Elsevier Science B.V. All rights reserved.