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 (TAE) 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 TBE buffers, the extrapolated mobilities at zero gel concentration increased gradually with decreasing DNA molecular weight, went through a maximum at approximately 60 bp, and then decreased again. The increase in the extrapolated mobilities with decreasing molecular weight observed for DNA fragments > or = 60 bp can be attributed to transient interactions between the migrating DNA molecules and the polyacrylamide gel fibers. If such interactions are eliminated by extrapolating the mobilities to both zero gel concentration and zero DNA molecular weight, the apparent free solution mobility of DNA is found to be 3.1 x 10(-4) cm2 V(-1) s(-1) in TAE buffer and 4.2 x 10(-4) cm2 V(-1) s(-1) in TBE buffer at 20 degrees C, reasonably close to the actual free solution mobilities measured in the same two buffers by capillary electrophoresis (N. C. Stellwagen et al., Biopolymers 1997, 42, 687-703). The significantly larger electrophoretic mobility observed in TBE buffer is most likely due to the formation of nonspecific, highly charged deoxyribose-borate complexes in this buffer medium. For DNA molecules < or = 60 bp in size, the decrease in the extrapolated mobilities with decreasing molecular weight parallels the decrease in their free solution mobilities observed by capillary electrophoresis. In agarose gels, the extrapolated mobilities of small DNA molecules at zero gel concentration appear to be independent of molecular weight. The apparent free solution mobilities are found to be (3.0 +/- 0.1) x 10(-4) cm2 V(-1) s(-1) in TAE buffer and (3.2 +/- 0.1) x 10(-4) cm2 V(-1) s(-1) in TBE buffer. The very similar mobilities observed in the two buffer media suggest that the borate ions in TBE buffer primarily form complexes with the galactose residues in the agarose gel fibers, rather than with the migrating DNA molecules, because of mass action effects. The formation of borate-agarose complexes, increasing the net negative charge of the agarose gel fibers, appears to be responsible for the markedly increased electroendosmotic flow observed in agarose gels cast and run in TBE buffer (N. C. Stellwagen, Electrophoresis 1992, 13, 601-603).
Strutz
K
K
Department of Biochemistry, University of Iowa, Iowa City, USA.
Stellwagen
N C
NC
D000085:Acetates; D000180:Acrylic Resins; D001888:Boric Acids; D002021:Buffers; D004247:DNA / Q000737:chemistry*; D004279:DNA, Viral / Q000737:chemistry; D004492:Edetic Acid; D004587:Electrophoresis, Agar Gel / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D012685:Sepharose; D012996:Solutions; D014325:Tromethamine
D000085:Acetates; D000180:Acrylic Resins; D001888:Boric Acids; D002021:Buffers; D004279:DNA, Viral; D012996:Solutions; C069591:Tris-borate-EDTA buffer; C016680:polyacrylamide gels; D014325:Tromethamine; D004247:DNA; D012685:Sepharose; D004492:Edetic Acid