Separation of long DNA fragments by capillary gel electrophoresis with laser-induced fluorescence detection.

Abstract

Long DNA fragments were separated by capillary gel electrophoresis with laser-induced fluorescence detection, and two approaches to obtaining high-resolution separation of DNA fragments were examined. One method achieved high resolution at the expense of detection time, while the other achieved both high resolution and fast detection. The former approach used a 300 cm long capillary with a gel concentration of 4% T and an electric field strength of 70 V/cm. The resolution limit in this electrophoresis was 800 bases with a resolution value of 0.5 for adjacent peaks. Under these conditions, the measurement time was 44 h. The latter approach used a 200 cm long capillary with a gel concentration of 3% T and an electric field strength of 170 V/cm. The resolution limit in this electrophoresis was 680 bases with a resolution value of 0.5 for adjacent peaks, and the measurement time was only 10 h. With both approaches, fragments with less than 600 bases were efficiently separated. The resolution values for adjacent peaks with less than 500 bases are greater than 1.0, and those for peaks with less than 100 bases are greater than 2.7. These approaches thus improve the accuracy of the base sequence determination.