Ultra-thin-layer agarose gel electrophoresis: II. Separation of DNA fragments on composite agarose–linear polymer matrices

The effect of hydrophilic linear polymer additives (non-cross-linked polyacrylamide, hydroxyethyl cellulose and polyethylene oxide) on the migration behavior of double stranded DNA molecules, ranging from 200–1000 base pairs, were studied in ultra-thin-layer agarose gel electrophoresis. The detection sensitivity was found to be less than 0.1 ng/band using To-Pro-3 fluorophore labeling and fiber optic bundle-based scanning detection system with a 640 nm red diode laser. Among the various polymers investigated, addition of linear polyacrylamide resulted in the best separation performance (steepest Ferguson plots), while composite gels with hydroxyethylcellulose and polyethylene oxide still exhibited adequate resolving power. Using the composite matrices of 1% agarose–linear polyacrylamide (0.5–3%), 1% agarose–hydroxyethylcellulose (0.2–1%) and 1% agarose–polyethylene oxide (0.2–1%), the mechanism of the separation was found to be in the Ogston sieving regime. Activation energy curves were also plotted based on the slopes of the Arrhenius plots of the various composite matrices, and exhibited decreasing characteristics for the agarose–linear polyacrylamide composite matrix and increasing characteristics for the agarose–hydroxyethylcellulose and agarose–polyethylene oxide composite matrices.