Vladislav Dolnik*, William A. Gurske, Ryan Smith and Allan Padua Molecular Dynamics, 928 East Arques Avenue, Sunnyvale, CA 94085, U.S.A. Telephone: +1 (408) 737-4810, Fax: +1 (408) 737-4808, e-mail: vlad.dolnik@am.apbiotech.com
		Introduction Capillary electrophoresis in sieving polymer solutions has become a popular tool for separation of biopolymers with separation of DNA sequencing fragments as the dominant application. Sieving polymers for DNA sequencing have been selected more or less empirically. Linear polyacrylamide (Ruiz-Martinez et al., 1993), polyethylene oxide (Fung and Yeung, 1995), hydroxyethyl cellulose (Bashkin et al., 1996), polydimethyl acrylamide (Madabhushi, 1998), polyvinyl pyrrolidone (Gao et al., 1999) are those used most frequently. According to a theory (Grossman and Soane, 1991), pore size in polymer solution depends on the polymer concentration only. The sieving performance, however, depends on the molecular mass of the polymer. This is because constraint release (Viovy et al., 1993) and network dissociation by migrating DNA fibers (Bae and Soane, 1991) are easier in polymers of lower molecular mass. The aim of this work was to give a background for a rational selection of sieving polymers in capillary electrophoresis in polymer solutions and comparison of sieving properties of various polymers independently of their concentration. Log-log mobility curve and inflection slope By calculating logarithm mobility for each DNA fragment and plotting them vs. logarithm number of DNA bases, a log-log mobility curve is obtained. Its shape depends on the concentration of the sieving polymer. As the concentration of the sieving polymer increases, the mobility of DNA decreases namely in the range of large DNAs.(Fig. 1). Figure 1. Log- log mobility curves of DNA in HEC, Mw 3 x 10^5. The slope of the mobility curve is related to selectivity (Dolnik and Gurske, 1999) and selectivity is the “engine” of the electrophoretic separation. (Giddings, 1969). The mobility curve slope reaches the minimum value at the inflection point of the mobility curve; it is the steepest point of the curve (Fig.
						(Fig. 4). The lines extrapolating the experimental data intercept the abscissa at the KD value. The plot is formally analogous to the Lineweaver-Burk plot used in enzymology and KD is formally analogous to the Michaelis constant. It expresses a concentration of polymer, at which the inflection slope equals to half of the inflection slope at the infinite concentration of polymer. It allows to describe the sieving properties of a polymer independently of its concentration. The lower KD, the better sieving polymer. Figure 4. Double reciprocal plot of inflection slope and concentration of hydroxyethyl cellulose. Intrinsic viscosity A physicochemical property of polymers has been sought that could be related to sieving properties of a polymer and that can be obtained by other methods than capillary electrophoresis. Intrinsic viscosity [h] was a candidate for such a constant. Intrinsic viscosity is obtained from viscosimetric measurements plotting ratio of difference between polymer viscosity and solvent viscosity, and polymer concentration Figure5. Determination of intrinsic viscosity of sieving polymers.
					2). Figure 2 Inflection point (black cross) and inflection slope (blue line) of a log- log mobility curve of DNA in 32 g/L HEC, Mw 3 x 10^5. As the concentration of the sieving polymer increases the log-log mobility curve becomes steeper and inflection slope values become more negative. At higher polymer concentrations, the decrease of the inflection slope is not as dramatic as it is at lower polymer concentrations (Fig. 3). According to the reptation model, the inflection slope cannot be smaller than –1. To find the minimum experimental value, a reciprocal plot has to be set up and the value at infinite polymer concentration is to be taken. Figure 3. Dependence of the inflection slope on concentration of sieving polymer. Double reciprocal plot A double reciprocal plot of the inflection slope and polymer concentration provides straight lines that intersect the ordinate at a point that is common for all polymers independently of molecular mass and chemical properties of sieving polymers
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