Do DNA gel electrophoretic mobilities extrapolate to the free‐solution mobility of DNA at zero gel concentration?

Abstract

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).

DNA separation by cholesterol-bearing pullulan nanogels

“…2. If the regression line was fitted for three data points at 25.0, 27.5, and 30.0 mg/ml, the y-intercept ͑3.82ϫ 10 −4 cm 2 V −1 s −1 ͒ became closer to the reported values [20][21][22][23] with a good correlation efficiency, r = 1.000. The other regression line for the rest of points also showed the similar value of slope and y-intercept, 4.16ϫ 10 −4 cm 2 V −1 s −1 .…”

“…However, the straight line, having a correlation coefficient of r = 0.974, intercepts the y-axis at a value of 3.02ϫ 10 −4 cm 2 V −1 s −1 , which does not agree well with the experimental data of free solution mobility reported elsewhere. [20][21][22][23] If we take a closer look around 30.0 mg/ml, a distinct mobility gap between 30.0 and 32.5 mg/ml of CHP could be found, which might be attributable to the phase transition of nanogels, as shown in Fig. 2.…”

DNA and Buffers: Are There Any Noninteracting, Neutral pH Buffers?

“…DNA-borate complexes are also observed when electrophoresis is carried out in polyacrylamide gels, using TBE as the running buffer (6,27). However, DNAborate complexes are not observed in agarose gels, because of the competition of the hydroxyl groups in the agarose gel fibers for the borate residues.…”

When can the Ogston-Morris-Rodbard-Chrambach model be applied to gel electrophoresis?

“…Strutz and Stellwagen [10] have recently also found experimentally that the mobility of small DNA extrapolates to the free solution mobility at zero gel concentration as predicted by Eq. (1).…”