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Separation of dsDNA in the presence of electroosmotic flow under discontinuous conditions
Author(s) -
Huang MingFeng,
Hsu ChienEn,
Tseng WeiLung,
Lin YuCheng,
Chang HuanTsung
Publication year - 2001
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/1522-2683(20017)22:11<2281::aid-elps2281>3.0.co;2-c
Subject(s) - capillary electrophoresis , ethidium bromide , polymer , haeiii , polyacrylamide , electrophoresis , chromatography , agarose , chemistry , capillary action , bromide , ethylene oxide , analytical chemistry (journal) , materials science , dna , polymer chemistry , organic chemistry , copolymer , polymerase chain reaction , biochemistry , restriction fragment length polymorphism , composite material , gene
Separations of φX‐174/ Hae III DNA restriction fragments have been performed in the presence of electroosmotic flow (EOF) using five different polymer solutions, including linear polyacrylamide (LPA), poly(ethylene oxide) (PEO), hydroxypropylcellulose (HPC), hydroxyethylcellulose (HEC), and agarose. During the separation, polymer solutions entered the capillary by EOF. When using LPA solutions, bulk EOF is small due to adsorption on the capillary wall. On the other hand, separation is faster and better for the large DNA fragments (> 872 base pairs, bp) using derivative celluloses and PEO solutions. Several approaches to optimum resolution and speed by controlling EOF and/or altering electrophoretic mobility of DNA have been developed, including (i) stepwise changes of ethidium bromide (0.5–5 µg/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 stepwise changes of ethidium bromide or voltage programming. Compared to PEO solutions, mixed polymer solutions prepared from PEO and HEC provide higher resolving power. Using a capillary filled with 600 m M TB buffers, pH 10.0, high‐speed (< 15 min) separation of DNA (pBR 322/ Hae III digest, pBR 328/ Bgl I digest and pBR 328/ Hin fI digest) has been achieved in 1.5% PEO.