Separation of double- and single-stranded DNA restriction fragments: capillary electrophoresis with polymer solutions under alkaline conditions.
Capillary electrophoresis in buffers containing hydroxyethyl cellulose (HEC) was used to separate double- and single-stranded DNA restriction fragments under neutral and alkaline conditions in epoxy-coated capillaries. It was found that better resolution was achieved using highly entangled HEC solutions for a narrow range of DNA fragment sizes, while lower resolution was obtained over a wide separation range using diluted HEC solutions. Optimal resolution of these DNA fragments was obtained using buffers containing 0.5% HEC at pH 11 with plate numbers exceeding 3 x 10(6) plates/m. It was also found that the diffusion coefficients and electrophoretic mobilities of DNA fragments decreased with increasing pH. This may indicate a more extended DNA conformation and, therefore, enhancement of transient entanglement coupling between DNA and HEC polymers under alkaline condition. At pH 12, ss-DNA were well separated in entangled HEC solutions; however, the resolution of ss-DNA was significantly decreased in diluted polymer solution.
D004247:DNA / Q000737:chemistry / Q000302:isolation & purification*; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000302:isolation & purification*; D019075:Electrophoresis, Capillary / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D009994:Osmolar Concentration; D011108:Polymers / Q000737:chemistry; D015203:Reproducibility of Results; D015183:Restriction Mapping; D012996:Solutions