Modification of the Electrokinetic Properties of Reversible Electrophoresis Gels for the Separation and Preparation of DNA: Addition of Linear Polymers

Abstract

The effect of adding linear polymers to a novel reversible electrophoretic was measured. Reversible gels are formed using the polyanionic carbohydrate polymer, gellan gum. Gellan gum forms strong stable gels in the presence of divalent cations or diamines. The gels are reversible (return to solution) by changing the ionic environment or pH. Gellan gum is an anionic polymer, and the electrophoresis gels have considerable electroosmotic flow (EOF) toward the negative electrode. We measured the EOF in gellan gum electrophoresis gels as a function of gel concentration, buffer composition, and linear polymer additive. The linear polymers used in this study were polyethylene oxide and hydroxyethyl cellulose. Both polymers reduced EOF in the gels, in a manner dependent on molecular weight. Polymers with high molecular weight were more effective at reducing EOF. The addition of polymers increased the resolution of low molecular weight DNA. Native gellan gum resolved DNA from approx 50,000 to 1000 bp. Addition of the polymers resolved DNA down to approx 50 bp, in some instances. The influence of the polymers on circular plasmid DNA was also investigated. Addition of high molecular weight polyethylene oxide reduced the electrophoretic mobility of the nicked circular form compared to the supercoiled form.

Improved separation of double‐stranded DNA fragments by capillary electrophoresis using poly(ethylene oxide) solution containing colloids

“…By taking the advantages of good dynamic coating ability of PDMA and good separation ability of linear polyacrylamide (LPA), a mixture of the two polymers was used for DNA separation [17]. Moreover, polymer solutions prepared from two different polymers have also been tested for DNA separation in the presence of electroosmotic flow (EOF) [18].…”

Characterization of Gellan Gum by Capillary Electrophoresis

“…Gellan gums are used as sieving media in gel electrophoresis. [16] Characterization of the gellan gums by electrophoresis is a lot scarcer. Free-solution capillary electrophoresis (CE) involves separation in a (fused-silica) capillary filled with a buffer solution (no stationary phase) and placed under high voltages (up to 30 kV).…”

DNA Electrophoresis in Gellan Gels. The Effect of Electroosmosis and Polymer Additives

“…The flow is oppositely directed to the motion of negatively charged macromolecules such as DNA and, therefore, tends to slow their migration and separation compared to conventional gels such as agarose. Cole and co-workers have found that the electroosmosis in the gels can be reduced by adding neutral polymers, for instance poly(ethylene glycol) (PEO). The addition of such polymers indeed increases the rate of DNA migration and thus separation, but the increase in DNA velocity was less than the decrease in electroosmotic flow, as measured independently.…”

“…Cole and co-workers have found that the electroosmosis in the gels can be reduced by adding neutral polymers, for instance poly(ethylene glycol) (PEO). The addition of such polymers indeed increases the rate of DNA migration and thus separation, but the increase in DNA velocity was less than the decrease in electroosmotic flow, as measured independently. This suggests that the added polymer has a second effect on the DNA migration that tends to counteract the indirect effect operating through the reduction of the electroosmotic flow.…”

“…This is accepted procedure in capillary electrophoresis and can be motivated in free solution on theoretical grounds, but the picture is less straightforward in gels because of how the gel network affects hydrodynamic interactions between polymer segments . For small solutes such as bromphenole blue, the subtraction procedure works well in gellan gels . With a large and flexible molecule such as DNA, on the other hand, the hydrodynamic and entanglement interactions with the gel fibers as well as with added PEO means that electrophoretic motion and a hydrodynamic flow may combine in a nonadditive manner.…”