The effects of polymer properties on DNA separations by capillary electrophoresis in uncross‐linked polymer solutions

Abstract

Low-viscosity, aqueous solutions of hydrophilic linear polymers have been shown to be useful for the separation of DNA restriction fragments by capillary electrophoresis (CE). However, the choice of polymer type, size, and concentration remains largely empirical, because the mechanism of high-field electrophoretic DNA separations in polymer solutions is not well understood. To assist in elucidating the mechanism of DNA separation, we experimentally investigated the effects of polymer properties such as stiffness (persistence length), average molecular mass, polydispersity, and hydrophilicity on the separation of DNA ranging from 72 bp to 23 kbp. This was accomplished by comparing the results of DNA separations obtained by counter-migration CE in dilute and semidilute solutions of linear polyacrylamide (PAA), hydroxyethyl-cellulose (HEC), and hydroxypropylcellulose (HPC) polymers of several different average molecular masses.

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.…”

“…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. …”

Identifying the Orientation of DNA Fragment in Recombinant Plasmid by Capillary Electrophoresis with a Non-Gel Sieving Solution

“…Recently, linear polymers have been popularly used in CE as the sieving matrix instead of the crosslinked gel, in terms of non-gel sieving or an entangled polymersolution CE, 13,14 due to its facilitation 15 of high separation speed, long read length and replaceable operation compared to CGE. Many polymers have proved to be useful, including linear polyacrylamide and its derivatives, 14,16,17 cellulose and its derivatives, 18,19 polyethene oxide 20,21 and agarose 22 etc. Currently, non-gel sieving CE is being extensively applied in DNA sequencing, characterizing the polymorphism of DNA, analyzing PCR products, gene diagnosis and therapy etc.…”