Electrophoresis In Polymer Solutions Research Articles

Divergent dispersion behavior of ssDNA fragments during microchip electrophoresis in pDMA and LPA entangled polymer networks.

Resolution of DNA fragments separated by electrophoresis in polymer solutions ("matrices") is determined by both the spacing between peaks and the width of the peaks. Prior research on the development of high-performance separation matrices has been focused primarily on optimizing DNA mobility and matrix selectivity, and gave less attention to peak broadening. Quantitative data are rare for peak broadening in systems in which high electric field strengths are used (>150 V/cm), which is surprising since capillary and microchip-based systems commonly run at these field strengths. Here, we report results for a study of band broadening behavior for ssDNA fragments on a glass microfluidic chip, for electric field strengths up to 320 V/cm. We compare dispersion coefficients obtained in a poly(N,N-dimethylacrylamide) (pDMA) separation matrix that was developed for chip-based DNA sequencing with a commercially available linear polyacrylamide (LPA) matrix commonly used in capillaries. Much larger DNA dispersion coefficients were measured in the LPA matrix as compared to the pDMA matrix, and the dependence of dispersion coefficient on DNA size and electric field strength were found to differ quite starkly in the two matrices. These observations lead us to propose that DNA migration mechanisms differ substantially in our custom pDMA matrix compared to the commercially available LPA matrix. We discuss the implications of these results in terms of developing optimal matrices for specific separation (microchip or capillary) platforms.

Anisotropic friction model of DNA electrophoresis in polymer solutions: Comparison with direct observations

AbstractThe electrophoresis of DNA chains in uncrosslinked polymer solutions with a Brownian dynamics simulation with an anisotropic friction tensor was analyzed. According to the degree of anisotropy, three types of migration behavior are obtained: fluctuation without or with periodicity between U‐shaped and compact conformations, or migration with linear conformation. We found good agreement between our simulation results and the direct observations of DNA by fluorescence microscopy. © 2003 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 41: 1316–1322, 2003

Simultaneous measurements of the electrophoretic mobility, diffusion coefficient and orientation of dsDNA during electrophoresis in polymer solutions.

We determined simultaneously the electrophoretic mobility, diffusion coefficient D and molecular orientation during electrophoresis of dsDNAs in polymer solutions ranging from the dilute to the semidilute regime. We established, for the first time, master scaling laws for the diffusion coefficient showing a universal behavior. A model found in the literature designed for the dilute regime allows, surprisingly, to describe the mobility data over the whole range of concentrations studied and at the same time the biased reptation with fluctuations (BRF) failed for the semidilute regime, even when constraint release of the network was taken into account. These quantitative determinations of D are of practical interest to evaluate band broadening during capillary electrophoresis and provide data for stimulating investigation of the physics of DNA electrophoretic motion.

Simultaneous measurements of the electrophoretic mobility, diffusion coefficient and orientation of dsDNA during electrophoresis in polymer solutions

We determined simultaneously the electrophoretic mobility, diffusion coefficient D and molecular orientation during electrophoresis of dsDNAs in polymer solutions ranging from the dilute to the semidilute regime. We established, for the first time, master scaling laws for the diffusion coefficient showing a universal behavior. A model found in the literature designed for the dilute regime allows, surprisingly, to describe the mobility data over the whole range of concentrations studied and at the same time the biased reptation with fluctuations (BRF) failed for the semidilute regime, even when constraint release of the network was taken into account. These quantitative determinations of D are of practical interest to evaluate band broadening during capillary electrophoresis and provide data for stimulating investigation of the physics of DNA electrophoretic motion.

Comparison of RNA, single-stranded DNA and double-stranded DNA behavior during capillary electrophoresis in semidilute polymer solutions

We present a study of the separation of RNA, single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) in semidilute linear hydroxyethylcellulose (HEC) solution. Our results strive to provide a better understanding of the mechanisms of nucleic acid migration during electrophoresis in polymer solutions under native and denaturing conditions. From a study of the dependence of mobility on chain length and applied electric field, we found that RNA and ssDNA show better separation and higher resolution over a larger range of sizes compared to dsDNA. In addition, RNA reptation without orientation extends to longer chain lengths in comparison to ssDNA, possibly as a result of different type of short-lived secondary structure formations. Such a comparative study between nucleic acid capillary electrophoresis helps to optimize RNA separation and provides better understanding of RNA migration mechanisms in semidilute polymer solutions under denaturing conditions.

Electric light scattering from single-stranded DNA in linear polyacrylamide solutions.

The electric light scattering (ELS) of ssDNA (calf thymus, 10 kbp, 55 micrograms/mL) in denaturing polyacrylamide (PAA) solutions was studied as a function of applied sinusoidal electric field and polymer concentration. Electric fields of strengths up to 300 V/cm and of frequencies between 100 and 5000 Hz were applied. It was found that the ELS effect increases with the field strength and decreases at high frequencies. The dependence of the ELS effect of ssDNA on polymer concentration passes through a maximum at 1% PAA. The relaxation times of decay of the ELS effect increase with increasing polymer concentrations. It was demonstrated that ELS is a useful method for investigation of ssDNA behavior in the course of pulse-field electrophoresis in polymer solutions.

Electric light scattering from single-stranded DNA in linear polyacrylamide solutions

The electric light scattering (ELS) of ssDNA (calf thymus, 10 kbp, 55 μg/mL) in denaturing polyacrylamide (PAA) solutions was studied as a function of applied sinusoidal electric field and polymer concentration. Electric fields of strengths up to 300 V/cm and of frequencies between 100 and 5000 Hz were applied. It was found that the ELS effect increases with the field strength and decreases at high frequencies. The dependence of the ELS effect of ssDNA on polymer concentration passes through a maximum at 1% PAA. The relaxation times of decay of the ELS effect increase with increasing polymer concentrations. It was demonstrated that ELS is a useful method for investigation of ssDNA behavior in the course of pulse-field electrophoresis in polymer solutions.

Electrohydrodynamically induced aggregation during constant and pulsed field capillary electrophoresis of DNA.

We present a study aimed at understanding the factors affecting the separation of large DNA molecules by capillary electrophoresis in polymer solutions. In a first series of experiments, a systematic study of the effect of operational parameters on the development of an electrohydrodynamic instability resulting in DNA aggregation and spurious peaks in the electropherograms is presented. The results are discussed in regard to a recent theory of electrohydrodynamic instabilities in macroion suspensions, recently proposed by Isambert et al. Overall, the results provide strong support to the theory. Some situations of interest for applications, and not explicitly considered in the theory, such as asymmetric field pulsing and the use of polymer additives in the buffer, were also considered. Furthermore, robust optimized protocols for high resolution separation of DNA in the range of 100 base pairs to 160 kilobase pairs, are proposed. As predicted by the model, it is shown that using a concentrated isoelectric buffer (histidine) strongly reduces aggregation as compared to the use of a conventional buffer at the same concentration, and allows separation of DNA from 100 bp to 160 kbp in less than 10 min. We also present a systematic study of the dependence of the mobility vs DNA size, pulse frequency, and field strength. The results are discussed with respect to the Biased Reptation with Fluctuations model and a good agreement is obtained. Copyright 1999 John Wiley & Sons, Inc.

A theory for the electrophoretic separation of DNA in polymer solutions.

We present a mathematical model of DNA capillary electrophoresis in polymer solutions based on nonentangling collisions between DNA and polymer molecules. Using videomicroscopy images of DNA migrating through polymer solutions, we propose a modified transient entanglement coupling mechanism for DNA separations that includes nonentangling DNA/polymer collisions. We show that a mathematical model based on individual nonentangling DNA/polymer collisions is able to predict the mobilities of DNA in solutions of low molecular mass polymer. We compare the model predictions to mobility data for separations of DNA in a range of concentrations for solutions of both hydroxypropylcellulose (Mw 100,000) and hydroxyethylcellulose (Mw 139,000). The model relies on one fitted parameter, the time constant for the interaction between the DNA and polymer molecules, which is based on the physical properties of DNA and polymers.

Protein Bandwidth in Gel Electrophoresis: A Primary Function of Migration Distance

By use of the fluorescent protein R-phycoerythrin, a number of factors have been investigated as potential sources of band broadening in agarose and polyacrylamide gels: temperature gradients, Joule heating, conductivity differences between analyte and buffer, electroendosmosis, microheterogeneity due to charge density differences, etc. It was found that none of these factors could be the major source of band broadening. Recently, it has been shown that diffusion is also not the predominant source of R-phycoerythrin band spreading in agarose gels and that the bandwidth depends linearly on migration distance and time (Yarmola, E.; Calabrese, P. P.; Chrambach, A.; Weiss, G. H. J. Phys. Chem. 1996, 101, 2381). The present data collected in agarose and polyacrylamide gels confirm the linear dependence of bandwidth on migration distance and describe the slope of this dependence as a function of electrophoretic conditions. Among all the factors studied that may impact on band spreading, only the interaction with the gel and microheterogeneity cannot be ruled out. This conclusion with regard to the mechanisms of band spreading agrees with that obtained recently by capillary electrophoresis in polymer solutions (Radko, S. P.; Weiss, G. H.; Chrambach, A. J. Chromatogr. A 1997, 781, 277−286).

Separation and isolation of subcellular‐size particles by electrophoresis in polymer solution using the commercial scanning apparatus

Electrophoresis of fluorescently labeled rat liver microsomes and polystyrene carboxylates of 10 and 30 nm radius was conducted in buffered 10-15% polyvinylpyrrolidone (M(r) = 10(6) solutions, using a horizontal gel electrophoresis apparatus with intermittent scanning of fluorescence (HPGE-1000, LabIntelligence). Banding, constant migration rates and Ferguson plots were obtained in these polymer solutions. The major microsome band detected by the automated scan was located visually on the gel by means of its fluorescein label and was isolated by volumetric withdrawal, recovery was monitored by scanning and ascertained to be near quantitative after three consecutive steps, in each of which 30 microL were withdrawn. This preparative method promises to be generally applicable to particles that are too large to enter into gels.

Differential electrophoretic behavior in aqueous polymer solutions of red blood cells from Alzheimer patients and from normal individuals

The recently reported phenomenon that red blood cells (RBC) from Alzheimer disease (AD) patients and normal individuals, which have identical electrophoretic mobilities (EPM) in phosphate-buffered saline (PBS), have different EPM in appropriately selected polymer solutions, has been further explored. Of a number of in vitro treatments to which AD and normal RBC were subjected prior to EPM measurements in bottom phase (from a dextran-poly(ethylene glycol) (PEG) aqueous phase system) only trypsin eliminated the difference. Thus, the differential polymer interaction between AD and normal RBC, thought to be the basis for their dissimilar EPM, can be abolished by appropriate proteolytic modification of the cell surfaces and suggests protein as a source of difference. Because young and old RBC from normal individuals, which have the same EPM in PBS, have different EPM in certain polymer solutions, and the RBC from AD patients have been reported to age abnormally, we also compared the young and old RBC subpopulations from these two sources. By the criterion of cell electrophoresis in polymer solutions the differences between AD and normal RBC and between young and old RBC are distinct. The EPM of AD and normal RBC differ in bottom phase or PEG but not in dextran solution; while the EPM of young and old RBC differ predominantly in dextran. We speculate that since the observed difference in EPM of RBC from AD patients and normals depends on protein(s) yet is anticoagulant-related (being obtained only when blood is collected in citrate or oxalate) it might be the result of an interaction (Ca 2+-mediated?) between the surfaces of these cells and protein component(s) of their respective, compositionally differing sera.

Molecular Sieving of Polystyrene Carboxylate of a Diameter up to 10 μm in Solutions of Uncrosslinked Polyacrylamide of Mr 5 × 10 6 Using Capillary Zone Electrophoresis

Capillary zone electrophoresis of polystyrene carboxylate (PSC) up to 10 μm in diameter in 0.1 to 0.9% solutions of uncrosslinked polyacrylamide (PA) of M r 5 × 10 6 demonstrates effective molecular sieving in that medium. The reduced mobility, however, is not constant in Tris-borate-EDTA (1× TBE) buffer containing the polymer but increases in proportion to the load of PSC and inversely to the concentration of PA unless the TBE concentration is increased 10-fold or 50 mM 3-[(3-cholamidopropyl) dimethylammonio]propanesulfonic acid is added to the solution or in the absence of PA. Both the Ferguson plots [log(mobility) vs PA concentration] of PSC obtained at various PSC loads and the effects of the dissociating conditions, high ionic strength and detergent, signify that the variable mobilities are those of different PSC aggregation states. Thus, only the fully associated or dissociated states of PSC provide mobilities that are constant independently of PSC load. The study shows that the information inherent in Ferguson plots regarding particle and fiber properties can be exploited for particles up to 10 μm in diameter by capillary zone electrophoresis in polymer solutions even if these particles form various aggregation states. Thus, such an analysis appears feasible in application to biologically relevant and functional complexes such as subcellular-sized particles or DNA-protein complexes, providing that these particles maintain their integrity and functionality under the selected electrophoretic conditions.

Cell partitioning in two-polymer aqueous phase systems and cell electrophoresis in aqueous polymer solutions. Human and rat young and old red blood cells

It has recently been found that electrophoresis in solutions of appropriately selected polymers in phosphate-buffered saline (PBS) can differentiate between some closely related cell populations which have identical electrophoretic mobilities (EPM) in PBS (e.g., human young and old red blood cells (RBC); RBC from Alzheimer patients and normal individuals). The EPM differences detected in polymer solutions are most likely a consequence of cell- and polymer-specific interactions. Aspects of the relation between the electrophoresis in aqueous polymer solutions of native and in vitro treated young and old RBC (from human and rat) and their partitioning in a charge-sensitive dextran-poly(ethylene glycol) (PEG) aqueous phase system (i.e., a system with a Donnan potential between the phases, top phase positive) have been examined further and are discussed. Unlike the behavior of RBC from Alzheimer patients and normal individuals in which an EPM difference can be detected in PEG solutions but not in dextran, differences in EPM between human young and old RBC are detectable in solutions of either polymer. Selected enzyme treatments of human young and old RBC or their fixation with aldehyde eliminates the EPM differences in dextran; while neuraminidase treatment or formaldehyde fixation of rat young and old RBC retains EPM differences in dextran between these cells. In these latter cases partitioning differences are also in evidence and are in the same direction as the cells' relative EPM (i.e., old RBC < young RBC). The earlier hypothesis that cell partitioning is ‘more sensitive’ than cell electrophoresis in detecting differences in surface charge between cells bears reexamination because human young and old RBC, which cannot be differentiated by single-tube partitioning in a charge-sensitive phase system, have different EPM in polymer solutions. The difference between these cells can be detected by partitioning but only by use of a multiple-extraction procedure. It is then found to be in a direction similar to the cells' relative EPM in dextran (i.e., human old RBC > young RBC ). Rat young and old RBC have different partitions (rat old RBC < young RBC) and different EPM (also rat old RBC < young RBC). Thus, while cell partitioning in a charge-sensitive dextran-PEG aqueous phase system and cell electrophoresis in polymer solution seem to reflect, at least with these cell subpopulations, qualitatively analogous differences in surface properties (in that increasing partitions and EPM are concomitant), there are instances in which either of these physical measurements discerns surface differences which escape detection by the other.

Cell partitioning in two-polymer aqueous phase systems and cell electrophoresis in aqueous polymer solutions red blood cells from different species

A correlation, with some exceptions, between the partitioning behavior of red blood cells (RBCs) from different species in charge-sensitive dextran-poly(ethylene glycol) (PEG) aqueous phase systems and their relative electrophoretic mobilities (EPMs) in phosphate-buffered saline (PBS) has previously been reported. This relationship has now been further probed by carrying out RBC electrophoresis in media ( i.e., dextran-rich bottom or PEG-rich top phases) more closely approximating the environment in which RBC partitioning takes place to see whether a better correlation would ensue. The ratios of viscosity-corrected EPMs of different species' RBCs in (diluted) dextran-rich or PEG-rich phases/EPMs of the respective species' RBCs in PBS differ for a number of species, and from each other, reflecting thereby differences in kind ( i.e., dextran or PEG) and nature of polymer interaction with these RBCs. There is a general tendency for EPMs in any of the tested media to correlate with both the cells' relative partition ratios as well as with their relative EPMs in one of the other media. However, examination of the behavior of different species' RBCs taken two species at a time indicates that their relative EPMs in any two suspending media or in one suspending medium and partitioning often differ. Thus, both the cell partition ratio and the cell EPMs obtained in polymer media must, at least in some cases, reflect surface properties other than or in addition to the charge reflected by EPM measurements in PBS or saline. Cell electrophoresis in polymer solutions thereby provides an additional parameter for discriminating between surface properties of certain closely related cell populations.

Comparative study of non-porous anion-exchange chromatography, capillary gel electrophoresis and capillary electrophoresis in polymer solutions in the separation of DNA restriction fragments

HPLC using a 100-mm column packed with a non-porous anion exchanger gave high-resolution separations of DNA restriction fragments up to 8000 base pairs (bp). High-resolution separation of DNA restriction fragments up to 12 000 bp was achieved using capillary gel electrophoresis (CGE) and capillary electrophoresis (CE) in entangled polymer solutions. These methods were compared with respect to their performance and efficiencies in the resolution of DNA restriction fragments. The resolving power of CGE is higher than those of the other techniques. The 50–100 bp resolution of DNA fragments up to 500 bp was realized using HPLC, 50 bp resolution using CE in entangled polymer solutions and 10 bp resolution using CGE. The plate number that was achieved with CGE of 3 · 10 6 plates/m was higher than those of HPLC of 1 · 10 6 and CE in polymer solutions of 7 · 10 5 plates/m. The advantages and limitations of HPLC and capillary electrophoretic techniques are discussed.

DNA electrophoresis in polymer solutions: Ogston sieving, reptation and constraint release.

The electrophoresis of long polyelectrolytes is considered theoretically, with special attention to duplex DNA. We first discuss quantitative approaches to determine unambiguously the entanglement properties of polymer solutions. Following an idea proposed by Grossman and Soane, we then assume that the "mesh" size of the solution plays the role of a dynamic "pore size" in order to apply theories for gel electrophoresis. In the framework of the Ogston model, we predict that duplex DNA up to 1 kb or more should be separable in dilute (i.e. nonentangled) solutions of high molecular weight polymers. In an entangled solution, and for DNA larger than the pore size, we use a recently developed fluctuation-reptation model to predict the range of sizes in which separation should be possible as a function of electric field E and pore size zeta b. For zeta b larger than the Kuhn length of DNA, we predict a separation up to a size N*scaling as E-1 zeta b-1. For zeta b smaller than the Kuhn length, two different regimes are expected. For small electric fields (typically of the order of 10 V/cm), N*should be proportional to E-1 zeta b-3/2, whereas for high electric fields such as encountered in capillary electrophoresis, we expect that N*is proportional to E-2/5 zeta b-12/5. These predictions are qualitatively different from earlier ones. Finally, we demonstrate that the finite lifetime of the "pores" in an entangled solution (as opposed to a gel) may lead to a new migration mechanism by constraint release, which is not size-dependent.(ABSTRACT TRUNCATED AT 250 WORDS)