Plant Genome III Conference Plant Genome III Conference
I will introduce capillary array electrophoresis (CAE) and show how have used this method to perform high-speed DNA sequencing, fragment sizing, microsatellite and STR analyses. In this technique, the DNA fragments are separated in gel-filled silica capillaries having an inside diameter of ~100 um and an outside diameter of ~300 um. The small diameter of these capillaries permits the application of high electric fields providing separations that are > 10-fold faster than slab gels. The capillaries are bundled to form a planar array and the fluorescently labeled DNA fragments are detected by using a laser-excited, confocal- fluorescence scanner. An additional advantage of CAE is that large numbers of samples can be easily loaded in parallel using standard electrokinetic injection techniques. Examples of DNA sequencing with 25-capillary arrays demonstrate sequencing rates of ~l0kb/hr.(1) Fragment sizing performed using capillary arrays filled with hydroxyethylcellulose solutions exhibit excellent resolution from 50 bp to ~10 kb in 20 minute runs.(2) The combination of confocal detection plus highly fluorescent intercalation dyes permits detection of DNA fragments present at pg/uL concentrations.(3) Two-color-labeling experiments using energy transfer PCR primers have demonstrated single base pair resolution of double-stranded THO1 allelic fragments in under 20 minutes.(4) Finally, a commercial prototype of a 48- capillary array system produced by Molecular Dynamics has been used to perform high-speed sizing of soybean microsatellite alleles obtained from Perry Creegans laboratory at the USDA.(5)