End-labeled free-solution electrophoresis of DNA

DNA is a free-draining polymer. This subtle but "unfortunate" property of highly charged polyelectrolytes makes it impossible to separate nucleic acids by free-flow electrophoresis. This is why one must typically use a sieving matrix, such as a gel or an entangled polymer solution, in order to obtain some electrophoretic size separation. An alternative approach consists of breaking the charge to friction balance of free-draining DNA molecules. This can be achieved by labeling the DNA with a large, uncharged molecule (essentially a hydrodynamic parachute, which we also call a drag-tag) prior to electrophoresis; the resulting methodology is called end-labeled free-solution electrophoresis (ELFSE). In this article, we review the development of ELFSE over the last decade. In particular, we examine the theoretical concepts used to predict the ultimate performance of ELFSE for single-stranded (ssDNA) sequencing, the experimental results showing that ELFSE can indeed overcome the free-draining issue raised above, and the technological advances that are needed to speed the development of competitive ELFSE-based sequencing and separation technologies. Finally, we also review the reverse process, called free-solution conjugate electrophoresis (FSCE), wherein uncharged polymers of different sizes can be analyzed using a short DNA molecule as an electrophoretic engine.     

Electrophoresis in the presence of gradients: I. Viscosity gradients

In many cases, the resolution provided by capillary electrophoresis systems approaches that predicted for diffusion-limited separations. Once all device-related sources of band broadening have been eliminated or minimized, only thermal diffusion remains. In principle, peaks can be sharpened using gradients of various system characteristics such as gel concentration, buffer viscosity and electric field. However, it is not clear whether this can actually increase the resolution of the system. In this article, we focus our attention on viscosity gradients and we examine both continuous and step-like variations. Our results indicate that the performance of electrophoretic systems cannot be improved by viscosity gradients. They may provide extra stacking, and thus improve the resolution, when the injection width is non-negligible. However, for the systems considered here, the best resolution is obtained when the viscosity is uniform and the stacking is entirely performed at injection. We conclude by discussing the link between these results, the fundamental laws of thermodynamics, the nature of the detection process and the importance of having nonlinear effects in nonuniform systems.     

Computational study of turbulent laminar patterns in couette flow

Turbulent-laminar patterns near transition are simulated in plane Couette flow using an extension of the minimal-flow-unit methodology. Computational domains are of minimal size in two directions but large in the third. The long direction can be tilted at any prescribed angle to the streamwise direction. Three types of patterned states are found and studied: periodic, localized, and intermittent. These correspond closely to observations in large-aspect-ratio experiments.     

Stable vortex-bright-soliton structures in two-component Bose-Einstein condensates

We report the numerical realization of robust two-component structures in 2D and 3D Bose-Einstein condensates with nontrivial topological charge in one component. We identify a stable symbiotic state in which a higher-dimensional bright soliton exists even in a homogeneous setting with defocusing interactions, due to the effective potential created by a stable vortex in the other component. The resulting vortex-bright-solitons, generalizations of the recently experimentally observed dark-bright solitons, are found to be very robust both in the homogeneous medium and in the presence of external confinement.     

Poloidal–toroidal decomposition in a finite cylinder. I: Influence matrices for the magnetohydrodynamic equations

The Navier–Stokes equations and magnetohydrodynamics equations are written in terms of poloidal and toroidal potentials in a finite cylinder. This formulation insures that the velocity and magnetic fields are divergence-free by construction, but leads to systems of partial differential equations of higher order, whose boundary conditions are coupled. The influence matrix technique is used to transform these systems into decoupled parabolic and elliptic problems. The magnetic field in the induction equation is matched to that in an exterior vacuum by means of the Dirichlet-to-Neumann mapping, thus eliminating the need to discretize the exterior. The influence matrix is scaled in order to attain an acceptable condition number.     

Free-solution electrophoresis of DNA modified with drag-tags at both ends

In end-labeled free-solution electrophoresis (ELFSE), DNA molecules are labeled with a frictional modifier or "drag-tag", allowing their size-based electrophoretic separation in free solution. Among the interesting observations from early work with dsDNA using streptavidin as a drag-tag was that the drag induced by including a streptavidin label at both ends was significantly more than double that from a single streptavidin (Heller, C. et al.., J. Chromatogr. A 1998, 806, 113-121). This finding was assumed to be in error, and subsequent work focused on experiments in which only a single drag-tag is appended to one end of the DNA molecule. Recent theoretical work (McCormick, L. C., Slater, G. W., Electrophoresis 2005, 26, 1659-1667) has examined the contribution of end-effects to the free-solution electrophoretic mobility of charged-uncharged polymer conjugates, reopening the question of enhanced drag from placing a drag-tag at both ends. In this study, this effect is investigated experimentally, using custom-synthesized ssDNA oligonucleotides allowing the attachment of drag-tags to one or both ends, as well as dsDNA PCR products generated with primers appropriate for the attachment of drag-tags at one or both ends. A range of sizes of drag-tags are used, including synthetic polypeptoid drag-tags as well as genetically engineered protein polymer drag-tags. The enhanced drag arising from labeling both ends has been confirmed, with 6-9% additional drag for the ssDNA and 10-23% additional drag for the dsDNA arising from labeling both ends than would be expected from simply doubling the size of the drag-tag at one end. The experimental results for ssDNA labeled at both ends are compared to the predictions of the recent theory of end-effects, with reasonably good quantitative agreement. These experimental findings demonstrate the feasibility of enhancing ELFSE separations by labeling both ends of the DNA molecule, leading to greater resolving power and a wider range of applications for this technique.     

Symmetry Breaking and Turbulence in Perturbed Plane Couette Flow

Perturbed plane Couette flow containing a thin spanwise-oriented ribbon undergoes a subcritical bifurcation at Re≈230 to a steady three-dimensional state containing streamwise vortices. This bifurcation is followed by several others giving rise to a fascinating series of stable and unstable steady states of different symmetries and wavelengths. First, the backwards-bifurcating branch reverses direction and becomes stable near Re≈200. Then the spanwise reflection symmetry is broken, leading to two asymmetric branches which are themselves destabilized at Re≈420. Above this Reynolds number, time evolution leads first to a metastable state whose spanwise wavelength is halved and then to complicated time-dependent behavior. These features are in agreement with experiments. Received 15 December 2001 and accepted 29 March 2002 Published online: 2 October 2002 Communicated by H.J.S. Fernando     

The molecular end effect and its critical impact on the behavior of charged-uncharged polymer conjugates during free-solution electrophoresis

Recently two novel techniques using free-solution electrophoresis to separate charged-uncharged polymer conjugates have proven successful: end-labeled free-solution electrophoresis (ELFSE) for DNA sequencing, and free-solution conjugate electrophoresis (FSCE) for molar mass profiling of uncharged polymers. The approach taken to analyze the experimental data was an extension of the theory of Long and co-workers (Long, D., Dobrynin, A. V., Rubinstein, M., Ajdari, A., J. Chem. Phys. 1998, 108, 1234-1244) for the electrophoresis of molecules with varying charge distributions. This theory also predicts that the ends of the polymers play a large role in determining the polymer's overall mobility; however, this aspect of the theory was neglected in previous work. Until now this "end effect" has, to the knowledge of the authors, not been recognized in experimental data. Through a careful investigation of the predicted end effect and a reanalysis of the experimental data, we demonstrate that indeed this effect critically impacts on the behavior of charged-uncharged polymer conjugates during electrophoresis. This work indicates that not only does the end effect need to be taken into account to avoid significant errors in data analysis, but also it provides novel system optimization approaches.     

Environmental noise and pure tone thresholds

In modern cities large populations are exposed to environmental noise due to transportation and industry, but empirical data are lacking as to the effect of this noise on pure tone thresholds. In this study the hearing of 10–12 year old children who had lived for the past 5–12 years in quiet parts of Sydney, Australia, was compared with that of children of the same age who had lived all of the past 5–12 years in noisy parts of the city. No difference was found in thresholds for pure tones from 500 to 6000 Hz.