High-speed parallel separation of DNA restriction fragments using capillary array electrophoresis.
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 approximately 20 min and detected with high sensitivity using a fluorescence detection system. The separations of phi X174/HaeIII fragments are performed on an array of seven 100-microns i.d., 350-microns 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 approximately 1 pg of DNA per band and separations can be detected injecting DNA samples as dilute as 0.1 ng/microliters. 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 approximately 100 capillary arrays is discussed.
Clark
S M
SM
Department of Chemistry, University of California, Berkeley 94720.
Mathies
R A
RA
D010584:Bacteriophage phi X 174 / Q000737:chemistry; D004279:DNA, Viral / Q000737:chemistry / Q000302:isolation & purification*; D015252:Deoxyribonucleases, Type II Site-Specific; D004586:Electrophoresis / Q000379:methods*
D004279:DNA, Viral; D015252:Deoxyribonucleases, Type II Site-Specific; C024403:GGCC-specific type II deoxyribonucleases