High-Speed Parallel Separation of DNA Restriction Fragments Using Capillary Array Electrophoresis

Abstract

A new method for performing high-speed, high-throughput sizing of DNA has been developed. Samples containing DNA restriction fragments between 70 and 10,000 base pairs in length are electrophoretically separated using capillary arrays in ∼20 min and detected with high sensitivity using a fluorescence detection system. The separations of ΦX174/HaeIII fragments are performed on an array of seven 100-μm i.d., 350-μm o.d. capillaries using replaceable hydroxyethyl-cellulose solutions as the sieving medium. The fragments are fluorescently labeled using ethidium bromide in the running buffer and detected with a laser-excited, confocal-fluorescence scanner. The limit of detection is ∼1 pg of DNA per band and separations can be detected injecting DNA samples as dilute as 0.1 ng/μl. Samples were electrokinetically injected from a cassette of microcentrifuge tubes using a format that will facilitate the application of capillary electrophoresis to separations in molecular biology. The feasibility of extending this technique to lower DNA concentrations and to ∼100 capillary arrays is discussed.