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G W Slater

Publications and source records attributed to G W Slater.

At least 19 recordsLinked to original sources
PubMed2001

Capillary electrophoretic separation of uncharged polymers using polyelectrolyte engines. Theoretical model.

We recently demonstrated that the molecular mass distribution of an uncharged polymer sample can be analyzed using free-solution capillary electrophoresis of DNA-polymer conjugates. In these conjugates, the DNA is providing the electromotive force while the uncharged polydisperse polymer chains of the sample retard the DNA engine with different amounts of hydrodynamic drag. Here we present a theoretical model of this new analytical method. We show that for the most favourable, diffusion-limited electrophoresis conditions, there is actually an optimal DNA size to achieve the separation of a given polymer sample. Moreover, we demonstrate that the effective friction coefficient of the polymer chains is related to the stiffness of the two polymers of the conjugate, thus offering a method to estimate the persistence length of the uncharged polymer through mobility measurements. Finally, we compare some of our predictions with available experimental results.

Biopolymers↗
PubMed2001

Molar mass profiling of synthetic polymers by free-solution capillary electrophoresis of DNA-polymer conjugates.

The molar mass distribution of a polymer sample is a critical determinant of its material properties and is generally analyzed by gel permeation chromatography or more recently, by MALDI-TOF mass spectrometry. We describe here a novel method for the determination of the degree of polymerization of polydisperse, uncharged, water-soluble polymers (e.g., poly(ethylene glycol) (PEG)), based upon single-monomer resolution of DNA-polymer conjugates by free-solution capillary electrophoresis. This is accomplished by end-on covalent conjugation of a polydisperse, uncharged polymer sample (PEG) to a monodisperse, fluorescently labeled DNA oligomer, followed by electrophoretic analysis. The monodisperse, charged DNA "engine" confers to each conjugate an equal amount of electromotive force, while the varying contour lengths of the uncharged, polydisperse polymers engender different amounts of hydrodynamic drag. The balance of electromotive and hydrodynamic forces enables rapid, high-resolution separation of the DNA-polymer conjugates as a function of the size of the uncharged PEG tail. This provides a profile of the molar mass distribution of the original polymer sample that can be detected by laser-induced fluorescence through excitation of the dye-labeled DNA. We call this method free solution conjugate electrophoresis (FSCE). Theory-based analysis of the resulting electrophoresis data allows precise calculation of the degree of polymerization of the PEG portion of each conjugate molecule. Knowledge of the molecular mass of the uncharged polymer's repeat unit allows for direct calculation of the molar mass averages as well as sample polydispersity index. The results of these analyses are strikingly reminiscent of MALDI-TOF spectra taken of the same PEG samples. PEG samples of 3.4-, 5-, and 20-kDa nominal average molar mass were analyzed by FSCE and MALDI-TOF; the values of the molar mass averages, Mw and Mn, typically agree to within 5%. Measurements and molar mass calculations are performed without any internal standards or calibration. Moreover, when DNA-polymer conjugate analysis is performed in a chip-based electrophoresis system, separation is complete in less than 13 min. FSCE offers an alternative to MALDI-TOF for the characterization of uncharged, water-soluble polymers that can be uniquely conjugated to DNA.

DNA↗
PubMed2001

An exactly solvable Ogston model of gel electrophoresis. VI. Towards a theory for macromolecules.

In this article, we present a generalized version of our lattice model of low-field gel electrophoresis that allows us to treat the case of macromolecules such as short linear or circular oligomers and semi-flexible rods. We show that free-solution electrophoresis problems can be seen as random walks in the conformational space of the analyte. For sufficiently small molecules, our mathematical approach provides exact mobilities. In a quenched gel-like environment, however, both conformational and positional degrees of freedom must be used, but exact solutions can also be obtained. As an example, we then investigate several two-dimensional model gels, as well as a simple channel system where we see evidence of entropic effects that cannot be captured by the traditional Ogston concept of free volume.

Electrophoresis, Agar Gel↗
PubMed2001

Diffusion coefficient of DNA molecules during free solution electrophoresis.

The free-draining properties of DNA normally make it impossible to separate nucleic acids by free-flow electrophoresis. However, little is known, either theoretically or experimentally, about the diffusion coefficient of DNA molecules during free-flow electrophoresis. In fact, many authors simply assume that the Nernst-Einstein relation between the mobility and the diffusion coefficient still holds under such conditions. In this paper, we present an experimental study of the diffusion coefficient of both ssDNA and dsDNA molecules during free-flow electrophoresis. Our results unequivocally show that a simplistic use of Nernst-Einstein's relation fails, and that the electric field actually has no effect on the thermal diffusion process. Finally, we compare the dependence of the diffusion coefficient upon DNA molecular size to results obtained previously by other groups and to Zimm's theory.

Algorithms↗
PubMed2001

An exactly solvable Ogston model of gel electrophoresis: VIII. Nonconducting gel fibers, curved field lines, and the Nernst-Einstein relation.

In this article, we examine the low-field electrophoretic migration of infinitely small analytes in dilute sieving media made of nonconducting gel fibers. Using an Ogston obstruction model, we show that the electrophoretic mobility is not affected by the presence of curved field lines. In other words, the Nernst-Einstein relation between the mobility and the diffusion coefficient is valid regardless of the electrical properties of the gel fibers. Although this finding may greatly simplify the development of obstruction models of electrophoretic sieving, it also represents a critical test for any analytical or computational approach.

Electrophoresis↗
PubMed2000

An exactly solvable Ogston model of gel electrophoresis. V. Attractive gel-analyte interactions and their effects on the Ferguson plot.

We examine the effect of attractive analyte-gel interactions within the framework of our recently developed lattice model of gel electrophoresis. We show that it is possible to take into account such interactions and still calculate exact mobilities for various analytes and gel structures. Our study then focuses on two main issues: (i) the effect of these interactions on the separation efficiency of the Ogston regime; and (ii) the presence of inflection points (changes of curvature) in Ferguson plots. We establish some general principles, and we describe the results for selected two- and three-dimensional model systems. Numerous practical problems, such as chiral separations and affinity electrophoresis, can be treated using this approach.

Chemical Phenomena↗
PubMed2000

Gel electrophoretic mobility of single-stranded DNA: the two reptation field-dependent factors.

The reptation model is the dominant theory in understanding the electrophoretic separation of single-stranded DNA molecules in gels or entangled polymer solutions. Recently, we showed that the Ogston and reptation regimes are separated by an entropic trapping regime at low field intensities. Here, we report the first comparison of the field-dependent part of the DNA mobility for both small and long reptating molecules. We show that both mobilities increase linearly with field intensity, with the mobility of the longer (comigrating) fragments increasing faster than that of the smaller ones. We compare our results to the predictions of the biased reptation model.

DNA, Single-Stranded↗
PubMed2000

Theory of DNA electrophoresis: a look at some current challenges.

Although electrophoresis is one of the basic methods of the modern molecular biology laboratory, new ideas are being suggested at an accelerated rate, in large part because of the pressing demands of the biomedical community. Although we now have, at least for some methods, a fairly good theoretical understanding of the physical mechanisms that lead to the observed peak spacings, widths and shapes, this knowledge is often too qualitative to be used to guide further technical developments and improvements. In this article, we review some selected elements of the current state of our theoretical ignorance, focusing mostly on DNA electrophoresis, and we offer several suggestions for further theoretical investigations.

Animals↗
PubMed1999

Separating DNA sequencing fragments without a sieving matrix.

The possibility of separating appropriately labeled DNA fragments using free-flow capillary electrophoresis was predicted a few years ago based on simple theoretical arguments. Free-flow separation of double-stranded DNA (dsDNA) fragments in the 100-1000 base range was later demonstrated using a streptavidin label. In this article, we now report that end-labeled free-flow electrophoresis (ELFSE) can also be used to sequence single-stranded DNA (ssDNA). The first 100 bases of a DNA sequencing reaction were read without any sieving matrix when fractionated streptavidin was added to the 5'-end of the ssDNA fragments. These separations required only 18 min and did not require coated capillaries. An analysis of the results indicates that sample injection, analyte-wall interactions and thermal diffusion are the limiting factors at this time. Extrapolating from our data, we predict that several hundred bases could be sequenced in less than 30 min with the proper conditions. ELFSE thus offers an attractive potential alternative to polymer solutions for DNA sequencing in capillaries and microchips.

Buffers↗
PubMed1999

Relaxation length of a polymer chain in a quenched disordered medium.

Using Monte Carlo simulations, we study the relaxation and short-time diffusion of polymer chains in two-dimensional periodic arrays of obstacles with random point defects. The displacement of the center of mass follows the anomalous scaling law r(c.m.)(t)(2)=4D(*)t(beta), with beta<1, for times t<t(SS), where t(SS) is the time required to attain the steady state. The relaxation of the autocorrelation function of the chain's end-to-end vector, on the other hand, is well described by the stretched exponential form C(t)=exp[-(t/tau(*))(alpha)], where 0<alpha</=1 and tau(*)<<t(SS). However, our results also obey the functional form C(r(c.m.))=exp(-[r(c.m.)/lambda](2)), implying the coupling alpha=beta even though these exponents vary widely from system to system. We thus propose that it is lambda, and not the traditional length (Dtau(*))(1/2), that is the relevant relaxation polymer length scale in disordered systems.

Journal Article↗
PubMed1998

The gel edge electric field gradients in denaturing polyacrylamide gel electrophoresis.

It has previously been shown that zones of higher electric field form close to the loading end of the gel during denaturing polyacrylamide gel electrophoresis. Here we show that the field can reach up to three times its normal mean value a few cm in front of the loading wells when 44.5 mM Tris-44.5 mM boric acid-1 mM EDTA is used as the gel buffer. We also demonstrate that this electric field gradient is mostly due to the difference in ion transference numbers at the gel/buffer interface caused by the high viscosity of the urea solution contained in the gel. This field gradient leads to increased band widths and forces us to redefine both the electrophoretic mobility and the mean field intensity. We discuss some methods that can be used to minimize the effects of this gradient.

Acrylic Resins↗
PubMed1998

Recent developments in DNA electrophoretic separations.

DNA electrophoresis is now a fairly mature technology. Nevertheless, as we approach the 21st century, new ideas are frequently suggested that could lead to a revolution for DNA sequencing and mapping. Here, we review some of the novel concepts that have been studied since ca. 1990. Our review focuses on new separation mechanisms, new sieving matrices and recent conceptual advances.

DNA↗
PubMed1998

An exactly solvable Ogston model of gel electrophoresis IV: sieving through periodic three-dimensional gels.

In this article, we extend our recently developed lattice model of gel electrophoresis to periodic three-dimensional gels made of either isolated obstacles or infinitely long fibers. Exact mobilities are calculated using a much improved numerical method that allows us to treat very large systems. A comparison of the exact mobilities and free available volumes indicates that the main assumption of the Ogston-Morris-Rodbard-Chrambach model (OMRCM), which postulates that the mobility (mu) of charged particles is directly related to the fractional gel volume available to them, is not valid. However, a study of the gel concentration and analyte size dependence of the zero-field mobility indicates that the OMRCM and the Ferguson plots can indeed be used to obtain useful, semi-quantitative information about the gel properties. A procedure to study more realistic three-dimensional gel systems is discussed.

Algorithms↗
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↗