Search PubMed⌕ Search

Biomedical subjects

G W Slater

Publications and source records attributed to G W Slater.

At least 37 records · Page 2Linked to original sources
PubMed1998

Trapping electrophoresis and ratchets: a theoretical study for DNA-protein complexes.

Recently, Griess and Serwer (1998. Biophys. J. 74:A71) showed that it was possible to use trapping electrophoresis and unbiased but asymmetrical electric field pulses to build a correlation ratchet that would allow the efficient separation of naked DNAs from identical DNAs that form a complex with a bulky object such as a protein. Here we present a theoretical investigation of this novel macromolecular separation process. We start by looking at the general features of this electrophoretic ratchet mechanism in the zero-frequency limit. We then examine the effects of finite frequencies on velocity and diffusion. Finally, we use the biased reptation model and computer simulations to understand the band-broadening processes. Our study establishes the main experimental regimes that can provide good resolution for specific applications.

Biophysical Phenomena↗
PubMed1996

Pulsed-field-trapping electrophoresis: a computer simulation study.

Experimental investigations have shown that adding a large, globular and neutral protein (such as streptavidin) at one end of the DNA fragments to be separated by gel electrophoresis strongly affects the dynamics of these molecules, leading to what is known as trapping electrophoresis (TE). In TE, the velocity decreases much more rapidly with DNA molecular size than under normal gel electrophoresis conditions, suggesting that TE may be used to increase the power of separation of polyacrylamide gel electrophoresis. Unfortunately, the bands are broader and fewer readable bands can fit on a single gel slab. Our previous theoretical study of TE also predicted the existence of long-lasting anomalous regimes where one cannot define a velocity or a diffusion constant. These secondary effects of trapping are related to the very broad distribution of detrapping times (the time needed to exit a trap). In order to increase the usefulness of TE, it has been suggested that pulsed fields may help the molecules exit traps more rapidly. In this article, we present a detailed numerical study of pulsed field TE. We conclude that simple pulsed fields alone may not be enough to increase the sequencing power of polyacrylamide TE because the rate of band broadening cannot be controlled. We also report the existence of anomalous regimes in the presence of pulsed fields, a factor that has been previously neglected in analytical models. Other approaches are also proposed.

Bacterial Proteins↗
PubMed1996

An exactly solvable Ogston model of gel electrophoresis: I. The role of the symmetry and randomness of the gel structure.

The Ogston-Morris-Rodbard-Chrambach model (OMRCM) of gel electrophoresis assumes that the mobility (mu) of charged particles is directly proportional to the fractional volume (f) of the gel that is available to them. Many authors have studied the fractional volume f in detail for various particle shapes, but the original assumption, that mu sf, has not been scrutinized seriously. In fact, this geometrical model of electrophoresis does not take into account the connectivity of the gel pores or the precise gel architecture. Recently (G. W. Slater and H. L. Guo, Electrophoresis 1995, 16, 11-15) we developed a Monte Carlo computer simulation algorithm to study the electrophoretic motion of simple particles in gels in the presence of fields of arbitrary strength. Our preliminary results indicated that the mobility and the fractional volume were not generally proportional to one another. In this article, we show how to calculate, in the limit where the field intensity is vanishingly small, the exact electrophoretic mobility of particles in any type of gel in two or more dimensions. Our results, presented here for some simple two-dimensional systems, indicate that a particle can have different electrophoretic mobilities in gels in which it has access to the same fractional available volume f. The curvature of the Ferguson plot is shown to be related to the symmetry and the degree of randomness that characterize the gel. We also demonstrate that the OMRCM is, in fact, a mean field approximation that corresponds to a uniform, annealed gel. We thus conclude that the relation between the electrophoretic mobility and the gel concentration (C) is a delicate function of the gel architecture, and that one needs more than the fractional volume f to fully characterize the transport properties of migrating particles in separation media. Exact relationships between the mobility mu and the gel concentration C are given for our model gels.

Animals↗
PubMed1996

An exactly solvable Ogston model of gel electrophoresis. II. Sieving through periodic gels.

Recently, we developed a lattice model to study the dynamics of particles being electrophoresed in gels (G. W. Slater, H. L. Guo, Electrophoresis 1995, 16, 11-15). In Part I of this series (G. W. Slater, H. L. Guo, Electrophoresis 1996, 17,977-988), we showed how to calculate the exact electrophoretic mobility of one-site particles in the limit where the electric field intensity E is vanishingly small. Since we can solve the model for arbitrary gel structures in two or more dimensions, we compared our results with those of the Ogston-Morris-Rodbard-Chrambach model (OMRCM) of gel electrophoresis, which assumes that the mobility (mu) of charged particles is directly proportional to the fractional gel volume (f) that is available to them. Our results and theoretical analysis indicated that the OMRCM is a mean-field approximation that can be useful as a rough guide; however, it generally misses the subtle sieving effects related to the correlations between the position of the obstacles in a given gel structure. In this paper (Part II) we study, for two-dimensional periodic gels, the exact relationships between the zero-field mobility mu and the gel concentration C for larger particle sizes. The fact that mu is a strong function of the particle size suggests that we can separate large particles using two-dimensional periodic gels (similar to those fabricated by W.D. Volkmuth and R.H. Austin, Nature 1992, 358, 600-602). We analyze our data using Ferguson-like plots and we show that one can indeed use a generalized retardation coefficient, K, to estimate the effective pore size aK and effective fiber size rK for these model gels. We conclude that the retardation coefficient is a useful concept to characterize a sieving structure even though it does not permit the inference of the exact gel structure.

Electrochemistry↗
PubMed1995

Ogston gel electrophoretic sieving: how is the fractional volume available to a particle related to its mobility and diffusion coefficient(s)?

The Ogston-Morris-Rodbard-Chrambach model (OMRCM) of gel electrophoresis assumes that the mobility mu of charged particles is proportional to the fractional volume (f) of the gel that is available to them. If the gel is random, as described by Ogston, the (semi-log) Ferguson plot is the method of choice for analyzing experimental data since it permits an estimate of the gel's mean pore size to be made. However, the Ferguson plot is rarely linear; this is usually "explained" by the deformation of the anisotropy of the particle, the nonrandom or variable architecture or the gel, or the onset of some other migration mechanism. Many authors have refined this model, but the original assumption that mu varied; is directly proportional to f has not been seriously examined. Also, the model says nothing of the effect of the field intensity, the connectivity of the gel pores, nor anything about the diffusion coefficient. We have developed a Monte-Carlo computer simulation algorithm to study the electrophoretic sieving of simple particles in gels. In this brief communication, we report important preliminary results which indicate that the basic assumptions of the OMRCM are wrong. We use a two-dimensional periodic gel since the OMRCM becomes trivial in this case. Our results show that the relationship between f and mu is not the one assumed by the OMRCM. Moreover, we find that the Einstein relation between the diffusion coefficient and the mobility is not valid. This is due to the fact that the particles do not have a uniform probability of visiting the various sites that are available to them. We thus conclude that the Ferguson plot is intrinsically nonlinear; the curvature of the plot is, in fact, related to the intensity of the electric field as well as to the degree of randomness of the gel fibers.

Algorithms↗
PubMed1995

Trapping gel electrophoresis of end-labeled DNA: an analytical model for mobility and diffusion.

As shown by Ulanovsky, Drouin and Gilbert (Nature 1990, 343, 190-192), the gel electrophoretic migration of DNA is severely reduced by steric trapping when streptavidin is attached to one end of the polyelectrolyte. We present a model that allows us to calculate both the mobility and the diffusion coefficient, hence the resolution factor of the resulting separation. We compare our results to those of Défontaines and Viovy (Electrophoresis 1993, 14, 8-17) and we show that the averages over the molecular conformations must be done carefully. We also show that trapping increases diffusion substantially and that this makes constant-field trapping electrophoresis incapable of increasing the number of bases read per sequencing run. Finally, we conclude that severe trapping may lead to highly anomalous transport behavior where one cannot define a velocity or a diffusion constant.

Bacterial Proteins↗
PubMed1995

Electrophoretic resolution versus fluctuations of the lateral dimensions of a capillary.

Because the local electrical resistance is inversely proportional to the local cross-section of a capillary, the intensity of the electric field varies along the migration path if the inner diameter of the capillary is not constant. Therefore, fluctuations of the lateral dimensions of a capillary can directly affect the net elution time as well as the peak width, and, hence the final resolution. In this article, we develop the theoretical framework for the study of such effects. We then examine the simple case where both the mobility and the diffusion coefficient are field-independent; in particular, we demonstrate that resolution can be severely reduced if the inner walls are not flat, and that optimal resolution is always obtained for perfectly flat walls. Generalized to ultrathin gels, our results clearly indicate that both random and systematic variations of the gel thickness can greatly affect the performance of the separation process. Acceptable degrees of flatness are estimated for both geometries. This study thus provides a quantitative understanding of the type of quality control one requires to obtain optimal results with capillaries and ultrathin gels.

Electrophoresis↗
PubMed1995

Diffusion, Joule heating, and band broadening in capillary gel electrophoresis of DNA.

We calculated the longitudinal and transverse diffusion coefficients for a DNA molecule undergoing gel electrophoresis in the limit where it is reptating through a dense polymer matrix. Our results indicate that both diffusion coefficients increase with the electric field intensity. The transverse and longitudinal diffusion coefficients are roughly equal for regular field intensities, but the former dominates for the high field intensities normally used for capillary gel electrophoresis (CE). This has important implications for the optimization of CE. Our results clearly show that the naive use of the zero-field diffusion constant, the Einstein relation or the longitudinal diffusion constant when calculating the contribution of the parabolic temperature profile to band broadening may lead to large overestimates under typical CE conditions. Finally, we show that the field-dependent diffusion coefficients may be responsible for the existence of an optimal field intensity for CE, even if Joule heating is neglected.

DNA↗
PubMed1995

Theory of capillary electrophoretic separations of DNA-polymer complexes.

Electrophoretic separation of DNA molecules normally requires the use of an anticonvection, sieving polymer matrix such as a gel or an entangled polymer solution. Recently, it has been suggested that free-solution separation could be achieved in a capillary if an electrically neutral, friction-generating molecule is attached to the DNA molecules before electrophoresis is carried out. The electrophoretic mobilities are then predicted to be very large and the resulting separation is expected to yield excellent resolution. The size-dependence of the electrophoretic mobility is attributed to longer DNA molecules pulling the neutral molecule with a larger electric force, thus eluting earlier than shorter DNA molecules. In this article, we focus on the particular case where one attaches an uncharged, flexible polymer to the end of the DNA. Our self-consistent model takes into account the deformation and the hydrodynamic resistance of the polymer in the flow. We find various regimes, depending on the intensity of the electric field and the length of the polymer. The most favorable conditions for high-resolution separation of DNA are described.

DNA↗
PubMed1994

Simulation of reduced band broadening during single-stranded DNA pulsed field electrophoresis in polyacrylamide gels.

Using a computer simulation algorithm based on the reptation model, we investigated the effects of pulsed field gel electrophoresis on the separation of single-stranded DNA molecules in denaturing polyacrylamide gels. Pulsed fields that combine two different field intensities were found to affect the orientation of the reptation tube as well as the electrophoretic velocity, the rate of band broadening, the plate height and the molecular length for which the minimum of mobility was found, in agreement with available experimental results. Due to "memory" effects, pulses alternating between the forward and backward directions can reduce the diffusion constant by many orders of magnitude. Pulses of identical polarity but different intensities reduce the diffusion because stretches of less-oriented DNA segments are conserved during the migration. We suggest that, for DNA sequencing, pulsed fields of fixed polarity should be used to reduce band broadening and not to overcome band inversion or suppress molecular orientation. We conclude that, using a low intensity, constant electric field will lead to smaller band widths than any pulsed field regime, in a similar length of experimental time, but with less complications.

Algorithms↗
PubMed1994

The biased reptation model of DNA gel electrophoresis: a user guide for constant field mobilities.

The biased reptation model of DNA gel electrophoresis is simple enough to allow one to obtain detailed analytical and numerical predictions for experimentally relevant situations. Although it is not always applicable for explaining experimental results, the biased reptation model is usually a good starting point for data analysis. Unfortunately, the model is often reported as being incapable of explaining experimental data because the users have not analyzed the data properly or because they attempted to use the model outside its expected range of applicability. This article presents a detailed practical guide to the model and its limitations, as well as a complete description of its predictions regarding the analysis of constant field mobilities.

DNA↗
PubMed1993

On the limits of near-equilibrium DNA gel electrophoretic sequencing.

High field intensities deeply affect the migration mechanisms of DNA in gels. The corresponding large electric forces lead to far-from-equilibrium transport properties such as the limiting mobility, increased diffusion and strong pulsed field responses. We present a theoretical study of the conditions required to retain a near-equilibrium reptation transport behaviour. We then examine the possibility of increasing both the field intensity and the gel concentration to sequence large single-stranded DNA molecules rapidly and efficiently. Finally, we discuss possible experimental approaches based on our theoretical predictions.

Base Sequence↗
PubMed1993

Exact behaviour of single-stranded DNA electrophoretic mobilities in polyacrylamide gels.

We demonstrate the existence of a gel edge effect where the velocity of the samples varies in the first and last centimetres of the gel. In spite of this effect, a differential method of velocity determination leads to exact mobility data. Further analysis against DNA length N and electric field E shows that the molecules always migrate according to an A/N+B(E) law, except in a limited range of E and N, whereas a 1/N1.6 entropic driven regime is found. Below a threshold electric field intensity B(E) varies as E2, in good agreement with the biased reptation mechanism, while at stronger electric fields intensities, B(E) stays constant. This second mechanism is not described by any actual theory, but might be attributed to a geometration-like mechanism. Implications of our findings in sequencing electrophoresis are discussed.

DNA, Single-Stranded↗
PubMed1992

Why can we not sequence thousands of DNA bases on a polyacrylamide gel?

Pulsed fields have been remarkably useful at extending the range of DNA molecular sizes that can be separated on agarose gels by controlling the field-induced molecular orientation that often limits the resolution of large molecules. Unfortunately, the same approach seems to be much less effective for DNA sequencing on polyacrylamide gels. We present an experimental and theoretical (modelling) study of DNA sequencing which shows that molecular orientation is indeed not the main limiting factor for sequencing devices that use moderate field intensities and polyacrylamide as a separating matrix. We examine the interplay between electric field intensity, molecular size and resolution, and we suggest different approaches to increase the resolution limit of standard and automated sequencing gels. The theoretical limits of high-field electrophoretic sequencing are also discussed. We conclude that new ideas will be needed to go beyond one kilobase.

Acrylic Resins↗
PubMed1990

Molecular detrapping and band narrowing with high frequency modulation of pulsed field electrophoresis.

In high electric fields, megabase DNA fragments are found to be trapped, i.e. to enter or migrate in the gel only very slowly, if at all, leading to very broad electrophoretic bands and loss of separation. As a consequence, low electric fields are usually used to separate these molecules by pulsed field electrophoretic methods. We report here that high-frequency pulses eliminate the molecular trapping found in continuous fields. When high frequency pulses are used to modulate the longer pulses used in pulsed field electrophoresis, narrower bands result, and higher fields can be used. We suggest that this is due to effects that occur on the length scale of a single pore.

Candida↗
PubMed1989

Generalized tube model of biased reptation for gel electrophoresis of DNA.

A theoretical analysis of the reptational motion of DNA in a gel that includes the effects of molecular fluctuations has been used to explain the main features found in experiments involving periodic inversion of the electric field. The resonance-like decrease of the electrophoretic mobility as a function of pulse duration is related to transient "undershoots" in the orientation of the molecule, in agreement with recent experimental data. These features arise from a delicate interplay of internal and center of mass motion of the molecules under pulsed field conditions, and are important for the separation of DNA molecules in the size range 0.2 to 10 million base pairs.

Chemical Phenomena↗
PubMed1989

The biased reptation model of DNA gel electrophoresis: mobility vs molecular size and gel concentration.

The biased reptation model provides a good framework for interpreting the results of continuous field DNA electrophoresis experiments performed in agarose gels. Here we discuss the main features of the mobility-molecular size and mobility-gel concentration diagrams as obtained from new extensive computer simulations of the model. Our aim is to suggest a global and coherent picture of this widely used yet poorly understood experimental technique, and to point out the areas where a systematic experimental study is still needed.

Computer Simulation↗