Characteristics of single-stranded DNA separation by capillary gel electrophoresis.

The optimization of electrophoretic conditions for fast separation of single stranded DNA by capillary gel electrophoresis was investigated. Mobilities and band broadening of the DNA fragments were measured for a 4% T, 5% C gel at 100-300 V/cm. The resolution of DNA peaks was evaluated for band spacing and band width. Band spacing decreased with increasing electric field strengths as well as DNA fragment size; resolution for large DNA fragments also decreased at higher electric field strengths. The measured mobilities in a 4% T, 5% C gel were analyzed by the reptation theory and compared with those in a 9% T, 0% C matrix. The reptation plots indicate that the onset of reptation in a 4% T, 5% C gel occurs at a DNA size of about 200 bases and about 150 bases in a 9% T, 0% C matrix. The 4% T, 5% C gel proved superior to the 9% T, 0% C matrix with respect to resolution and analysis time. The optimum electric field strength and migration distance for specific DNA fragment length (200, 400 and 700 bases) in the shortest time were calculated from band spacing and the measured band width for the 4% T, 5% C gel at Rs = 0.5. Under optimized conditions, analysis of DNA fragments up to 200, 400 and 700 bases could be carried out in about 5, 50, and 230 min, respectively.