Insights from a New Analytical Electrophoresis Apparatus

Abstract

Charge is a fundamental property of macromolecules that is inextricably linked to their structure, solubility, stability, and interactions. Progress has been made on the theoretical and structural aspects of these relationships. However, for several reasons, charge is difficult to measure in solution. Consequently, there is a lack of experimental data that, independent of other macro-ion properties, determines the effective charge. To overcome this problem, novel instrumentation and methods are being developed in our laboratory. Described here is an analytical electrophoresis apparatus that permits both the measurement of electrophoretic mobilities and the determination of steady-state electrophoresis concentration distributions. The latter provides a different-perspective on the processes that influence macro-ion behavior in an electric field. In addition, the apparatus permits the determination of diffusion coefficients either from boundary spreading during transport or from the decay of a concentration gradient. All of these determinations can be made with a single, 8-microL sample in a variety of solvents, thus providing unique insights into the charge properties of a macro-ion. Presented here is a progress report about this emerging technology, including the description of a prototype apparatus and examples of its use with a DNA oligonucleotide.

Direct force measurements on DNA in a solid-state nanopore

“…Extensive modelling was required to convert the measured DNA mobility into effective charge 16,17,[19][20][21][22] 23 , which is remarkable given the complex interplay between hydrodynamics and electrical charges in the screening layer of the DNA. Our data may provide a basis for developing a microscopic theory of the dynamics of ions on DNA.…”

The Length Dependence of Translational Diffusion, Free Solution Electrophoretic Mobility, and Electrophoretic Tether Force of Rigid Rod-Like Model Duplex DNA

“…There remains little doubt that such interactions occur as the different mobilities of DNA in Tris-acetate and Tris-borate buffers under otherwise identical experimental conditions demonstrate (Stellwagen et al, 1997(Stellwagen et al, , 2000. With regard to past modeling, it should be mentioned that the electrophoretic mobility of 20-bp DNA in 0.11 M KCl at 20°C measured by the technique of membrane-confined analytical electrophoresis (Laue et al, 1996) are fairly well explained by continuum modeling. The model mobilities are ϳ4 -6% higher than experiment under these conditions (Allison and Mazur, 1998;Mazur et al, 2001).…”

“…Condition III is chosen to make contact with recent capillary electrophoresis studies of Hoagland et al (1999) on long duplex DNA. The membrane-confined capillary electrophoresis studies of Laue et al (1996) on short DNAs were carried out under conditions very similar to condition V. Conditions II-V also provide insight into the effect of small-ion mo-bility and ionic strength on transport properties of DNA models. Finally, condition VI is carried out in 0.2 M KCl at 1.3°C ( ϭ 1.711 cp, ⑀ 0 ϭ 87.49).…”

“…The most straightforward applications of BE modeling to electrokinetic transport is to free solution electrophoresis (Laue et al, 1996;Stellwagen et al, 1997;Rasmusson and Akerman, 1998;Hoagland et al, 1999) and its application to DNA of moderate length has been described in the present work. Its extension to the very useful technique of gel electrophoresis (Calladine et al, 1991;Gurrieri et al, 1999) is currently underway.…”

Electrokinetic techniques applied to electrochemical DNA biosensors

“…The electrophoretic force purely derived of the electrokinetic phenomena on NA can be alternatively expressed as F EP 5 q eff N DNA E, where q eff is the effective charge per base pair and N DNA is the number of base pairs of the DNA molecule. Currently, q eff is estimated to be between 0.066 and 0.30e À per base pair in plasmids and double-stranded DNA (dsDNA) [4][5][6] and it will be a function of the ionic strength.…”