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 polyac rylamide, hydroxyethyl cellulose and polyethylene oxide) on the migration b ehavior of double stranded DNA molecules, ranging from 200-1000 base pairs, were studied in ultra-thin-layer agarose gel electrophoresis. The detectio n sensitivity was found to be less than 0.1 ng/band using To-Pro-3 fluoroph ore labeling and fiber optic bundle-based scanning detection system with a 640 nm red diode laser. Among the various polymers investigated, addition o f linear polyacrylamide resulted in the best separation performance (steepe st Ferguson plots), while composite gels with hydroxyethylcellulose and pol yethylene oxide still exhibited adequate resolving power. Using the composi te matrices of 1% agarose-linear polyacrylamide (0.5-3%), 1% agarose-hydrox yethylcellulose (0.2-1%) and 1% agarose-polyethylene oxide (0.2-1%), the me chanism of the separation was found to be in the Ogston sieving regime. Act ivation energy curves were also plotted based on the slopes of the Arrheniu s plots of the various composite matrices, and exhibited decreasing charact eristics for the agarose-linear polyacrylamide composite matrix and increas ing characteristics for the agarose-hydroxyethylcellulose and agarose-polye thylene oxide composite matrices. (C) 2000 Elsevier Science B.V. All rights reserved.