Electrophoretic dynamics of large DNA stars in polymer solutions and gels (original) (raw)
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Electrophoretic mobility of linear and star-branched DNA in semidilute polymer solutions
Electrophoresis, 2006
Electrophoresis of large linear T2 (162 kbp) and 3-arm star-branched (NArm = 48.5 kbp) DNA in linear polyacrylamide (LPA) solutions above the overlap concentration c* has been investigated using a fluorescence visualization technique that allows both the conformation and mobility μ of the DNA to be determined. LPA solutions of moderate polydispersity index (PI ∼ 1.7–2.1) and variable polymer molecular weight Mw (0.59–2.05 MDa) are used as the sieving media. In unentangled semidilute solutions (c*<c<ce), we find that the conformational dynamics of linear and star-branched DNA in electric fields are strikingly different; the former migrating in predominantly U- or I-shaped conformations, depending on electric field strength E, and the latter migrating in a squid-like profile with the star-arms outstretched in the direction opposite to E and dragging the branch point through the sieving medium. Despite these visual differences, μ for linear and star-branched DNA of comparable size are found to be nearly identical in semidilute, unentangled LPA solutions. For LPA concentrations above the entanglement threshold (c>ce), the conformation of migrating linear and star-shaped DNA manifest only subtle changes from their unentangled solution features, but μ for the stars decreases strongly with increasing LPA concentration and molecular weight, while μ for linear DNA becomes nearly independent of c and Mw. These findings are discussed in the context of current theories for electrophoresis of large polyelectrolytes.
Electrophoresis, 2004
We study the effect of matrix chain molecular weight Mw and concentration c on the electrophoretic mobility μ of large linear and star-like, branched DNA in polymer solutions. Polyethylene oxide (PEO) with narrow molecular weight distributions form the main focus of this study. For PEO concentrations ranging from one half the overlap concentration, c*, to 3c*, the effective drag coefficient, ζ , satisfies the following approximate scaling relationship, ζ . Here, μ0 is the electrophoretic mobility in free solution. While the concentration dependence is consistent with predictions from the transient entanglement coupling (TEC) model, the molecular weight dependence is significantly weaker. Although a similar dependence of mobility on Mw can be predicted when nonentangling collisions are the dominant source of drag, a model based on these collisions alone cannot reproduce the experimental observations. We also find that the architecture of large DNA does not affect either the concentration dependence or molecular weight dependence of the electrophoretic mobility.
Topological effects on the electrophoretic mobility of rigid rodlike DNA in polyacrylamide gels
Biopolymers, 2003
The electrophoretic migration of rigid rodlike DNA structures with well defined topologies has been investigated in polyacrylamide (PA) hydrogels prepared by copolymerization of acrylamide and N, NЈ-methylenebisacrylamide. Previous studies have reported structural and dynamic characteristics of linear and branched DNA during electrophoresis in PA gels using a variety of experimental parameters. However, a thorough investigation aimed at establishing specific relationships between topological features of rigid rodlike DNA structures and their electrophoretic behavior is still needed. In order to study these topological effects on mobility, an intensive examination of the electrophoretic mobility of small linear and starlike DNA was performed. A series of model DNA structures with well-defined branched topologies were synthesized with varying molecular parameters, such as number of arms surrounding the branch point and arm length. The electrophoretic mobility of these structures was then contrasted with a series of data obtained using linear DNA of comparable molecular size. When large DNA stars (M Ն 60 bp) were compared with linear DNA of identical molecular weight, the Ferguson plots were quite different. However, small DNA stars (24-32 bp) and linear analogues had identical Ferguson plots. This indicates that a different motional mode or greater interaction with the gel exists for the larger DNA stars. When the total molecular weight of the DNA stars was held constant and the number of arms varied, the Ferguson plots for all the stars were identical. Additionally, a critical pore size was reached when the ratio of linear DNA mobility to star DNA mobility increased dramatically. Thus, while the incorporation of a single branch point can produce a large reduction in mobility, above a critical molecular size, the incorporation of additional branch points does not appear to provide further reduction in mobility. This finding is consistent with the transport properties of large synthetic star polymers, where a large reduction in their diffusion coefficient is observed when a single branch is added. When additional arms are incorporated, large synthetic stars do not display an appreciable further reduction in diffusion coefficient. The effect of arm length on mobility for rigid rod DNA stars was also studied. For four-arm DNA stars, the mobility was found to scale as
Single Chain Observation on Collapse Transition in Giant DNA Induced by Negatively-Charged Polymer
Biochemical and Biophysical Research Communications, 1998
and lambda). In the present study, we examined the Poly-glutamic acid, a polyanion, has been shown to effect of crowding with anionic polymer on the compacinduce the all-or-none collapse transition in individual tion of DNA by observing individual DNAs with fluoduplex DNAs from an elongated coil into a collapsed rescence microscopy (11,12). With this technique, we compact state, through the observation of single can monitor changes in the higher-order structure of chains by fluorescence microscopy. This experimental single long DNAs in an aqueous environment. We finding is discussed in relation to the change in the adapted poly-L-glutamic acid (p-Glu) as a negatively higher-order structure of DNAs in the cytoplasmic charged polymer. In this paper, we avoid to use term space induced by a change in the concentration of of ''condensation'', in order to make clear the fact that polyanions, such as mRNA, as a possible route for we have focused on the event with respect to single feedback in gene expression.
Analytical …, 2007
We have studied the effects of polymer molar mass and concentration on the electrophoretic migration modalities of individual molecules of DNA in LPA, HEC, and PEO solutions via epifluorescent videomicroscopy. While both transient entanglement coupling (TEC) and reptation have been studied in the past, the transition between them has not. Understanding this transition will allow for polymer network properties to be optimized to enhance the speed and resolution of DNA separations in microfluidic devices. Near the overlap threshold concentration, C*, TEC is the dominant observed mode of DNA migration, and the observation frequency of TEC increases with increasing polymer molar mass. As polymer concentration is increased, observed TEC events reduce to zero while DNA reptation events become the only detected mechanism. Individual DNA molecules undergoing both migration mechanisms were counted in solutions of varying polymer molar masses and concentrations and were plotted against a dimensionless polymer concentration, C/C*. The data for LPA reduce to form universal curves with a sharp increase in DNA reptation at ∼6.5C*. Analogous transition concentrations for PEO and HEC were observed at 5C* and 3.5C*, respectively, reflecting the different physical properties of these polymers. This transition correlates closely with the polymer network entanglement concentration, C e , as measured by rheological techniques. The electrophoretic mobility of λ-DNA in LPA polymer solutions was also measured and shows how a balance can be struck between DNA resolution and separation speed by choosing the desired prevalence of DNA reptation.
Electrophoresis, 1994
Using capillary electrophoresis, large DNA molecules (2.0-23.1 kbp) may be rapidly separated in ultradilute polymer solutions (< 0.002% w/w) under a high-voltage, steady field (265 V/cm). At this polymer concentration, the separation mechanism appears to be significantly different from that postulated to occur in crosslinked gels. Based on experimental results obtained with DNA restriction fragments and with negatively charged latex microspheres, we conclude that the Ogston and reptation models typically used to describe gel electrophoresis are not appropriate for DNA separations in such dilute polymer solutions. Electrophoresis experiments employing solutions of both small and large hydroxyethyl cellulose polymers highlight the importance of polymer length and concentration for the optimum resolution of DNA fragments varying in size from 72 bp to 23.1 kbp. A transient entanglement coupling mechanism for DNA separation in dilute polymer solutions is developed, which suggests that there is no a priori upper size limit to DNA that can be separated by capillary electrophoresis in a constant field.
Journal of Molecular Biology, 1999
DNA heteroduplexes as models for slipped strand DNA have been analyzed by polyacrylamide gel migration and atomic force microscopy (AFM). All heteroduplexes containing one hairpin or loop have reduced electrophoretic mobilities compared with that expected for their molecular weights. The retarded gel mobility correlates with the formation of a sharp kink detected by AFM. Increasing the hairpin length from 7 bp to 50 bp results in a monotonous decrease in gel mobility of heteroduplexes. This secondary retardation effect appears to depend only on the hairpin size since the AFM data show no dependence of the kink angle on the hairpin length. Heteroduplex isomers with a loop or hairpin in opposite strands migrate with distinct mobilities. Analysis of gel migration of heteroduplexes with altered hairpin orientations as well as of truncated heteroduplexes indicates that the difference in mobility is due to an inherent curvature in one of the long arms. This is con®rmed by the endto-end distance measurements from AFM images. In addition, signi®cant variation of the end-to-end distances is consistent with a dynamic structure of heteroduplexes at the three-way junction. Double heteroduplexes containing one hairpin in each of the complementary strands also separate in a gel as two isomers. Their appearance in AFM showed a complicated pattern of¯at representations of the three-dimensional structure and may indicate a certain degree of interaction between complementary parts of the hairpins that are several helical turns apart.
Influence of branch length asymmetry on the electrophoretic mobility of rigid rod-like DNA
Electrophoresis, 2004
The electrophoretic mobility of three-arm asymmetric star DNA molecules, produced by incorporating a short DNA branch at the midpoint of rigid-rod linear DNA fragments, is investigated in polyacrylamide gels. We determine how long the added branch must be to separate asymmetric star DNA from linear DNA with the same total molecular weight. This work focuses on two different geometric progressions of small DNA molecules. First, branches of increasing length were introduced at the center of a linear DNA fragment of constant length. At a given gel concentration, we find that relatively small branch lengths are enough to cause a detectable reduction in electrophoretic mobility. The second geometric progression starts with a small branch on a linear DNA fragment. As the length of this branch is increased, the DNA backbone length is decreased such that the total molar mass of the molecule remains constant. The branch length was then increased until the asymmetric branched molecule becomes a symmetric three-arm star polymer, allowing the effect of molecular topology on mobility to be studied independent of size effects. DNA molecules with very short branches have a mobility smaller than linear DNA of identical molar mass. The reason for this change in mobility when branching is introduced is not known, however, we explore two possible explanations in this article. (i) The branched DNA could have a greater interaction with the gel than linear DNA, causing it to move slower; (ii) the linear DNA could have modes of motion or access to pores that are unavailable to the branched DNA.
Anomalous gel migration of DNA oligomers containing multiple conformational junctions
Biochemistry, 1990
We have previously shown that a short 16 base pair D N A oligomer can accommodate a B-Z conformational junction [Sheardy, R. D., & Winkle, S. A. (1989) Biochemistry 28, 720-7251, Results from 'H N M R studies indicated that only three base pairs were involved in the junction and that one of these base pairs was highly distorted. Being interested in the nature of this distortion, we constructed D N A oligomers which have the potential to contain multiple B-Z junctions for polyacrylamide electrophoretic