Separation of Double- and Single-Stranded DNA Restriction Fragments: Capillary Electrophoresis with Polymer Solutions under Alkaline Conditions
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Abstract
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.
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