Free-solution electrophoresis, of DNA

Capillary Electrophoresis in Analytical Biotechnology, Righetti, P.G., Ed. CRC Press Boca Raton, FL, 1996. Capillary Electrophoresis—Theory and Practice. New Directions in Organic and Biological Chemistry Series Camilleri, P., Ed. CRC Press Boca Raton, FL, 1997. Heller, C. Slater, G.W. Mayer, P. Dovichi, N. Pinto, D. Viovy, J.-L. Drouin, G. Free-solution electrophoresis of DNA. J. Chromatogr., A 1998, 806 (1), 113-221. 

Free-solution electrophoresis of DNA that has been conjugated to a friction-perturbing entity, which we call a drag-tag, was not feasible until after the development of CE in the 1990s. Free-solution electrophoresis generates a significant amount of heat, which must be dissipated. The narrow channels used in CE have diameters in the order of 25-100 p,m, which efficiently remove this Joule heat. Free-solution conjugate electrophoresis (FSCE) was first examined quantitatively in 1994 by Mayer... 

Meagher, R. J. et al. Free-solution electrophoresis of DNA modified with drag-tags at both ends. Electrophoresis 27,1702-1712 (2006). 

McCormick, L.C. and G.W. Slater, Molecular deformation and free-solution electrophoresis of DNA-uncharged polymer conjugates at high field strengths Theoretical predictions. Part 1 Hydrodynamic segregation. Electrophoresis, 2007 28(4) 674-682. 

Stellwagen, NC, Apparent Pore Size of Polyacrylamide Gels Comparison of Gels Cast and Run in Tris-acetate-EDTA and Tris-borate-EDTA Buffers, Electrophoresis 19, 1542, 1998. Stellwagen, NC Gelfi, C Righetti, PG, The Free Solution Mobility of DNA, Biopolymers 42, 687, 1997. 

FIGURE 12.15 Graph of size resolution factor as a function of the number of ssDNA bases that are sequenced. When the size resolution factor is < 1, single-base resolution of ssDNA fragments is possible. The scaled EOF mobility is the EOF mobility divided by the free-solution mobility of DNA. Therefore, the read-length is predicted to be the longest when the EOF mobihty exactly equals the free-solution mobility of DNA. (Reprinted with permission from McCormick, L. C. and Slater, G W. Electrophoresis 27,1693-1701, 2006.)... 

Mayer, R, Slater, G. W., and Drouin, G. Theory of DNA-sequencing using free-solution electrophoresis of protein-DNA complexes. Analytical Chemistry 66, 1777-1780 (1994). 

Electrophoresis experiments in glass tubes were reported as early as in the nineteenth century, but the first real breakthrough occurred in the first half of the twentieth century when the Swedish chemist Arne Tiselius applied free-solution electrophoresis—i.e moving boundary—to serum protein analysis, for which he later received the 1937 Nobel Prize [2], In less than two decades, just after the striking scientific discovery of the double-helical structure of DNA by Watson and Crick in 1953 [3] and the following unveiling of the genetic code, electrophoresis became a standard and indispensable tool in the field of modern... 

Nathakarnkitkool S, Oefner PJ, Bartsch G, Chin MA, Bonn GK (1992) High resolution capillary electrophoretic analysis of DNA in free solution. Electrophoresis 13 18-31. 

Size-based separations of homogeneous polyelectrolytes, such as DNA, are not possible in free solution electrophoresis [159]. This is due to the proportionality of the friction hydrodynamic force and total charge of the molecule to its length. The friction hydrodynamic forces exerted on the free-drained polymer coil while it moves as well as the accelerating electrostatic force both increase proportionally with the addition of a nucleotide to the chain. This is why one must typically use a sieving media, such as a gel or an entangled polymer solution, to obtain size-based separations of DNA using electrophoresis. 

The first iteration of FSCE theory predicted the potential sequencing of up to 2000 bases in less than 1 h under perfect (diffusion-limited) conditions using a drag-tag with an a between 100 and 200. The initial theory, however, assumed that /u.(M) and the diffusion coefficient D M) eould be related using the Nernst-Einstein equation, which it turns out is not a valid assumption under free-solution electrophoresis conditions. This assumption caused Mayer to overestimate the potential performance of DNA sequencing by FSCE. Eurther development of the theory with a eorreetion for this error led to the following equation that estimates the maximum number of bases that ean be sequenced by a drag-tag with a specified a-value ... 

Vreeland, W. N. et al. Molar mass profiling of synthetic polymers by free-solution capillary electrophoresis of DNA-polymer conjugates. AnaZyficaZ Chemistry 13, 1795-1803 (2001). 

Nkodo, A. E. et al. Diffusion coefficient of DNA molecules during free solution electrophoresis. Electrophoresis 22, 2424—2432 (2001). 

The above discussion makes clear that colloidal particles and polyelectrolytes cannot be separated by size during free solution electrophoresis. As a result, many electrophoretic size separations for chemistry and biology are performed in gels. The precision afforded by microfabrication has led to miniaturized version of these classic protocols, as well as a number of novel separation techniques that differ distinctly from the separation principles prevailing in gels [5]. In order to best understand the current research in microfluidic separations of colloids and poly electrolytes, in particular the important apphcations to protein and DNA separations, it is important to first understand the physics of gel electrophoresis. 

Nkodo, A., Gamier, J., Tinland, B., Ren, H., Desraisseaux, C., McCormick, L., Drouin, G., and Slater, G., 2001. Diffusion coefficient of DNA molecules during free solution electrophoresis. Electrophoresis, 22, 2424-2432. 

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