Capillary electrophoretic separation of dsDNA under nonuniform electric fields.
Improved sensitivity for the analysis of DNA by capillary electrophoresis has been achieved, based on simultaneous increases in optical path length and injection volume. To increase the optical path length, bubble cells with diameters ranging from 150 to 450 microm have been fabricated and tested. In terms of resolution and sensitivity, a bubble cell of 300 microm diameter is appropriate when using 75-microm capillaries. To allow greater injection volumes, we performed on-line concentration of DNA in the presence of electroosmotic flow (EOF) using 2.0% poly(ethylene oxide) (PEO). With a 300-microm bubble cell, a 170-fold improvement in the sensitivity for the 89-bp fragment has been accomplished when injecting about 0.33 microL DNA. In the presence of the bubble cell, the resolution for the large fragments improves while that for the small ones (<124 base pair) decreases. The effect of bubble cells was further investigated by conducting DNA separation in the absence of EOF, showing that improvements in resolution are mainly due to increased migration differences when DNA migrated at low electric field strengths in the bubble region. We have suggested that such an effect is more profound using shorter capillaries, leading to complete separation of phiX 174 RF DNA-Hae III digest in 2 min.
Department of Chemistry, National Taiwan University, Section 4, Roosevelt Road, Taipei, R.O.C.
Yang-Wei
YW
Lin
Chih-Ching
CC
Huang
Huan-Tsung
HT
Chang
D004247:DNA / Q000737:chemistry / Q000302:isolation & purification*; D004560:Electricity; D019075:Electrophoresis, Capillary / Q000379:methods*; D012680:Sensitivity and Specificity
D004247:DNA