Pulsed field capillary electrophoresis of nucleic acids using variable pulse duration protocols.

Abstract

Pulsed field capillary electrophoresis using variable pulse duration protocols employs a field-modulation waveform consisting of changing pulse durations and amplitudes. The use of this technique allows for optimal, or near optimal separation conditions for a wide range of DNA fragment lengths to be employed in one electrophoretic separation. Optimal separation conditions depend upon the nucleic acid fragment length and shape and its interactions with the linear polymer matrix. The mobilities and resolution of fragments shorter than the interaction length are amplitude dependent, but not pulse duration dependent. It is observed that the mobilities and resolution of fragments longer than the interaction length are both amplitude and pulse duration dependent. Mobility and resolution effects in pulsed field capillary electrophoresis of nucleic acids in entangled linear polyacrylamide matrices are illustrated with examples of separations of $\phi$X174/HaeIII, 1 kilobase ladder, and $\lambda$DNA/Hind/III fragments. Variable pulse duration protocols are also applied to nucleic acid separations in dilute and ultra-dilute hydroxyethyl cellulose (HEC) solutions. More bands are detected in these separations than there are DNA fragments in the sample. The extra bands are real, not artifacts of the separation technique, and are observed over a wide range of DNA, intercalator, and dilute HEC concentrations. These bands are a result of DNA aggregation and matrix polymer aggregation during electrophoresis. Groups of adduct mobilities are associated with a certain DNA fragment lengths. This is illustrated with examples of $\lambda$DNA/HindIII digest fragment separations in dilute and ultra-dilute HEC solutions. Pulsed field capillary electrophoresis is also used for the analysis of single stranded polysaccharides in dilute HEC. Dextran sulfates were employed as model analytes in the separations with indirect fluorescence as the detection method. HEC aggregation during electrophoresis results in unstable and irreproducible backgrounds making it impossible to identify analyte peaks in the dextran separations.