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

Abstract

The polysacharide gellan forms hydrogels if a divalent ion such as calcium is added at millimolar concentrations. The gel can be reversed to solution by adding EDTA, which makes it a promising candidate for preparative electrophoretic separation of biomolecules. We have studied the electrophoretic migration of double-stranded T4 DNA (164 kilobase pairs) in gellan gels by velocity measurements and by linear dichroism spectroscopy studies of the DNA coil conformation during the migration. The gels either contained 0.3% of high molecular weight (5 × 10 6 ) poly(ethylene oxide) (PEO), to suppress the electroosmosis induced by the negative charges of the gellan polymer, or were free of such added polymer and therefore exhibited an electroosmotic flow which is opposite to the DNA migration. In both cases, the viral DNA migrate in an oscillatory manner between stretched and coiled states, because it becomes entangled with the gellan gel fibers. In the stretched state of the cycle, the molecules are substantially aligned with the field. As the field is turned off, the alignment relaxes first by a rapid (seconds) stretch relaxation along the aligned path in the gel, followed by a slower (minutes) end-on type of motion to the equilibrium isotropic coil state. The added PEO has two effects on the DNA migration. An indirect effect is that the quenched electroosmosis leads to a stronger stretching of the DNA and to shorter cycle period times because the ends of the molecules move faster in the absence of the counter flow. A direct effect is that the PEO itself retards the DNA motion, most likely because of a combination of hydrodynamic interactions and entanglement effects. The net result of these two opposing PEO effects is that the center-of-mass velocity of the DNA increases by a factor of about 2 upon addition of PEO.

Cytoplasmic transport and nuclear import of plasmid DNA

“…In our studies, fluorophore-labeled PNAs (PNA Bio Inc, Newbury Park, California, USA) were bound to plasmids in a triplex structure that is stable in cells and serum for several days [ 66 , 108 – 110 ], whereas in the study by Dhanoya and colleagues [ 106 ], plasmids were labeled with the intercalating YOYO-1 dye. While YOYO-1 binds to DNA with high affinity and is extremely stable for electrophoresis at low salt concentrations, at physiological salt concentrations, dissociation of YOYO-1 can occur within seconds to minutes [ 111 , 112 ]. It has been found that labeling methods for plasmids can have profound effects on the DNA in terms of transcriptional activity or subcellular localization.…”

Electroosmosis with step changes in zeta potential in microchannels

“…For electrophoresis, some researchers attempted to uniformly suppress EOF2, 3 while others took advantage of the EOF4, 5 to improve performance. In either case, the surface should be electrokinetically homogeneous in order to avoid convective sample dispersion, which lowers separation efficiency.…”

Groove-binding unsymmetrical cyanine dyes for staining of DNA: dissociation rates in free solution and electrophoresis gels

“…The dimeric YOYO has been used successfully to monitor the electrophoretic motion of DNA in agarose gels for tens of minutes in Tris±borate buffers with an approximate concentration of 50 mM of monovalent ions (6). However, if the salt concentration was increased to 200 mM (monovalent ions) or if divalent calcium ions were used at a concentration of 5 mM, the YOYO dye was observed to dissociate within seconds (7), which rendered microscopy observations impossible. This is a crucial limitation when salt concentration is being used as a parameter to modulate electrostatic interactions by screening, or when divalent ions are used for instance as gelling agents in reversible gels for preparative electrophoresis (7).…”

“…However, if the salt concentration was increased to 200 mM (monovalent ions) or if divalent calcium ions were used at a concentration of 5 mM, the YOYO dye was observed to dissociate within seconds (7), which rendered microscopy observations impossible. This is a crucial limitation when salt concentration is being used as a parameter to modulate electrostatic interactions by screening, or when divalent ions are used for instance as gelling agents in reversible gels for preparative electrophoresis (7). An additional potential disadvantage of cyanine dyes based on the YO and TO chromophores is that they bind to DNA by intercalation.…”