Separation of dsDNA in the presence of electroosmotic flow under discontinuous conditions

Separations of phiX-174/HaeIII DNA restriction fragments have been performe d in the presence of electroosmotic flow (EOF) using five different polymer solutions, including linear polyacrylamide, (LPA), poly(ethylene oxide) (P EO), hydroxypropylcellulose (HPC), hydroxyethylcellulose (HEC), and agarose . During the separation, polymer solutions entered the capillary by EOF. Wh en using LPA solutions, bulk EOF is small due to adsorption on the capillar y wall. On the other hand, separation is faster and better for the large DN A fragments (> 872 base pairs, bp) using derivative celluloses and PEO solu tions. Several approaches to optimum resolution and speed by controlling EO F and/or altering electrophoretic mobility of DNA have been developed, incl uding (i) stepwise changes of ethidium bromide (0.5-5 mug/mL), (ii) voltage programming (125-375 V/cm), (iii) use of mixed polymer solutions, and (iv) use of high concentrations of Tris-borate (TB) buffers. The DNA fragments ranging from 434 to 653 bp that were not separated using 2% PEO (8 000 000) under isocratic conditions have been completely resolved by either stepwis e changes of ethidium bromide or voltage programming. Compared to PEO solut ions, mixed polymer solutions prepared from PEO and HEC provide higher reso lving power. Using a capillary filled with 600 mm TB buffers, pH 10.0, high -speed (< 15 min) separation of DNA (pBR 322/HaeIII digest, pBR 328/Bg/l di gest and pBR 328/Hinfl digest) has been achieved in 1.5% PEO.