Capillary electrophoresis of DNA in uncross-linked polymer solutions

Abstract

The electrophoretic separation of DNA molecules is usually performed in thin slabs of agarose or polyacrylamide gel. However, DNA separations can be achieved more rapidly and efficiently within a microbore fused silica capillary filled with an uncrosslinked polymer solution. An early assumption was that the mechanism of DNA separation in polymer solutionxapillary electrophoresis (PS-CE) is the same as that postulated to occur in slab gel electrophoresis, i.e., that entangled polymer chains form a network of "pores" through which the DNA migrates. However, we have demonstrated that large DNA restriction fragments (2.0-23.1 kbp) can be separated by CE in extremely dilute polymer solutions, which contain as little as 6 parts per million (0.0006% (w/w)l of uncrosslinked hydroxyethyl cellulose (HEC) polymers. In such extremely dilute HEC solutions, far below the measured polymer entanglement threshold concentration, pore-based models of DNA electrophoresis do not apply. We propose a transient entanglement coupling mechanism for the electrophoretic separation of DNA in uncrosslinked polymer solutions, which is based on physical polymer/DNA interactions. 0 1996 John Wiley & Sons, Inc.

Evaluation of capillaries with different inner coatings for DNA analysis using dilute polymer solutions by capillary electrophoresis

“…Some possible causes for the differences in our results with the uncoated capillary, in comparison with the data from literature, 1,[17][18][19] can be mentioned here. The dimensions of the capillary used by Barron et al 1 were different from ours (51 µm i.d.…”

“…The resolution of the fragments with different sizes, however, was not possible in this capillary, and it was not possible to reproduce the historical data from literature. 1,[17][18][19] The behavior expected in this capillary was first the arrival of the larger fragments at the detector, followed by the smaller ones. With the presence of electroosmotic flow, the bigger molecules are carried more easily to the detector, because of their slower mobilities.…”

“…Because of this, the discrimination of the solute by size no longer exists, so electrophoretic separation ability based on molecular size is lost. 13,16 The separation of DNA fragments in polymers below the entanglement threshold (c*) was first accomplished by Barron et al, 17,18 proving that the formation of a physical net by the polymer molecules is not essential for the solute separation. This fact showed that the Ogston and the reptation theories are not enough to explain the DNA migration mechanism in physical gels.…”

“…The more fibers one fragment drags, the lower is its mobility over time; and the larger the fragment, the greater is the probability of its interaction with the polymers in solution. 1,[17][18][19] …”

“…This intercalator modifies DNA structure, reducing its flexibility. Since the basis of DNA separation in diluted polymers (the "transient entanglement coupling" theory) is the interaction of DNA molecules with the gel fibers, 1,[17][18][19] this structure modification in DNA may also change the separation mechanism. …”