Separation of DNA sequencing fragments using an automated capillary electrophoresis instrument.
Rapid, high-resolution separation of DNA sequencing fragments by capillary gel electrophoresis using an automated, commercially available instrument is presented. The effect of column lengths and electric field strength on the resolution of sequencing fragments as well as the sensitivity of laser-induced fluorescence (LIF) detection was investigated. Using a short capillary of 20 cm length, which results in a U-shape of the capillary in the capillary cartridge, very high separation efficiency, up to 17 x 10(6) theoretical plates per m, is obtained. Analysis of the band broadening factors revealed that the resolution on the short column is predominantly determined by axial diffusion and to a minor extent by detection zone width. Presumably due to the coiling of longer capillaries in the capillary cartridge, increasing the capillary length does not increase the separation efficiency as predicted for diffusion-limited separation. The concentration limit of detection (signal-to-noise ratio = 2) is 0.2 x 10(-12) M of fluorescein-labeled oligonucleotide primer under the separating conditions for DNA sequencing samples. Increasing the electric field strength from 100 to 175 V/cm improved resolution and at the same time approximately doubled the sequencing speed. Fragments up to 500 nucleotides in length are resolved in less than 50 min.
Karger
A E
AE
Deutsches Krebsforschungszentrum, Heidelberg, Germany. a.karger@dkfz.heidel erg.de
D001322:Autoanalysis / Q000295:instrumentation*; D001483:Base Sequence; D004247:DNA / Q000737:chemistry*; D004574:Electromagnetic Fields; D019075:Electrophoresis, Capillary / Q000295:instrumentation*; D004591:Electrophoresis, Polyacrylamide Gel / Q000295:instrumentation*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009841:Oligonucleotides / Q000302:isolation & purification*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013696:Temperature
D009841:Oligonucleotides; D004247:DNA