DO DNA GEL-ELECTROPHORETIC MOBILITIES EXTRAPOLATE TO THE FREE-SOLUTION MOBILITY OF DNA AT ZERO GEL CONCENTRATION

The electrophoresis of small DNA fragments has been measured in dilute agarose and polyacrylamide gels cast and run in Tris-acetate-EDTA (TA E) and Tris-borate-EDTA (TBE) buffers. Ferguson plots were constructed to extrapolate the mobilities to zero gel concentration and estimate the free solution mobility of DNA. In polyacrylamide gels, in both TAE and TEE buffers, the extrapolated mobilities at zero gel concentratio n increased gradually with decreasing DNA molecular weight, went throu gh a maximum at similar to 60 bp, and then decreased again. The increa se in the extrapolated mobilities with decreasing molecular weight obs erved for DNA fragments greater than or equal to 60 bp can be attribut ed to transient interactions between the migrating DNA molecules and t he polyacrylamide gel fibers. If such interactions are eliminated by e xtrapolating the mobilities to both zero gel concentration and zero DN A molecular weight, the apparent free solution mobility of DNA is foun d to be 3.1 x 10(-4) cm(2)V(-1)s(-1) in TAE buffer and 4.2 x 10(-4) cm (2)V(-1)s(-1) in TBE buffer at 20 degrees C, reasonably close to the a ctual free solution mobilities measured in the same two buffers by cap illary electrophoresis (N. C. Stellwagen et al., Biopolymers 1997, 42, 687-703). The significantly larger electrophoretic mobility observed in TEE buffer is most likely due to the formation of nonspecific, high ly charged deoxyribose-borate complexes in this buffer medium. For DNA molecules less than or equal to 60 bp in size, the decrease in the ex trapolated mobilities with decreasing molecular weight parallels the d ecrease in their free solution mobilities observed by capillary electr ophoresis. In agarose gels, the extrapolated mobilities of small DNA m olecules at zero gel concentration appear to be independent of molecul ar weight. The apparent free solution mobilities are found to be (3.0 +/- 0.1) x 10(-4) cm(2)V(-1)s(-1) in TAE buffer and (3.2 +/- 0.1) x 10 (-4) cm(2)V(-1)s(-1) in TBE buffer. The very similar mobilities observ ed in the two buffer media suggest that the berate ions in TEE buffer primarily form complexes with the galactose residues in the agarose ge l fibers, rather than with the migrating DNA molecules, because of mas s action effects. The formation of borate-agarose complexes, increasin g the net negative charge of the agarose gel fibers, appears to be res ponsible for the markedly increased electroendosmotic flow observed in agarose gels cast and run in TBE buffer.