Estimation of polyacrylamide gel pore size from Ferguson plots of linear DNA fragments. II. Comparison of gels with different crosslinker concentrations, added agarose and added linear polyacrylamide.

The mobilities of various DNA fragments in two normally migrating molecular weight ladders were studied in polyacrylamide gels containing different concentrations of the crosslinker N,N'-methylenebisacrylamide (Bis). The acrylamide concentration ranged from 2.5-10.5%T (w/v); the Bis concentration ranged from 0.5-10%C (w/w), with respect to total acrylamide. Ferguson plots were constructed for each of the DNA fragments in gels of each composition. The Ferguson plots of the different multimers in each molecular weight ladder were nearly parallel in gels containing 0.5-3%C, converged close to a common intercept at zero gel concentration in gels containing 4%C, and crossed at approximately 1.5%T in gels containing 5 and 10%C. If the mobilities observed for the different DNA fragments at zero gel concentration were also extrapolated to zero DNA molecular weight, a common limiting mobility was observed in gels of all crosslinker concentrations. This limiting mobility was approximately equal to the free solution mobility of DNA. The effective pore radius of each gel was estimated from Ferguson plots based on relative mobilities, using the mobility of the smallest DNA fragment in each molecular weight ladder as the reference mobility. The calculated gel pore radii ranged from 142 nm to 19 nm, respectively, for gels containing 4.6%T, 1.5%C, and 10.5%T, 5 or 10%C. These pore radii are an order of magnitude larger than previously accepted values, but are consistent with scanning electron microscope measurements (Rüchel, R., et al., J. Chromatogr. 1978, 42, 77-90).(ABSTRACT TRUNCATED AT 250 WORDS)     

Free mobility determination by electrophoresis in polyacrylamide containing agarose at a nonrestrictive concentration

In the determination of the free mobility, related to the surface net charge, by quantitative gel electrophoresis, the previous arbitrary extrapolation of Ferguson plots from the lowest gel concentrations that give a mechanically stable gel to 0% T has recently been replaced by measurement of mobilities across that concentration range, using the addition of 0.5% agarose to polyacrylamide at the various low concentrations in application to a DNA fragment 155 bp in size (Orbán, L. et al., in preparation). The present study applies that approach to several proteins and DNA fragments smaller than 1300 bp, using 0.4% agarose in polyacrylamide gels of varying concentration. The intercepts of the plots with the mobility axis provide experimental data by which the free mobility in polyacrylamide gel electrophoresis can be estimated for molecules not significantly retarded in their migration at the agarose concentration admixed to polyacrylamide. Across the gel concentration range below 3% T, in the presence of agarose, the Ferguson plots of proteins and DNA fragments are convex. It was shown by mass spectrometry that this convex curvature of the plots in the mixed polymer is not significantly due to low polymerization efficiency in the concentration range of liquid polyacrylamide (below 3%T).     

Concave Ferguson plots of DNA fragments and convex Ferguson plots of bacteriophages: evaluation of molecular and fiber properties, using desktop computers.

A desktop computer program evaluating physical properties of DNA and bacteriophages is presented. The analysis is based on data obtained from capillary and submarine-type agarose electrophoresis. Native molecular/particle properties and properties of the gel (or polymer) medium can be derived from electrophoresis at several gel concentrations. This is done conveniently by a computerized evaluation of the semi-logarithmic plot of mobility vs. gel concentration, designated the Ferguson plot. In application to most proteins, this plot is linear and computer programs exist to evaluate it. However, nonlinear Ferguson plots have assumed great importance in view of the fact that the plots are concave for DNA. Similarly, convex plots are important since they prevail in the electrophoresis of large particles in agarose. The computer program reported here is the first to (i) address concave Ferguson plots and (ii) allow for the evaluation of both cases using a desktop computer. Program ELPHOFIT version 2.0, a Macintosh application, is available upon request.     

The electric field dependence of DNA mobilities in agarose gels: a reinvestigation

The electric field dependence of the electrophoretic mobility of linear DNA fragments in agarose gels was reinvestigated in order to correct the observed mobilities for the different temperatures actually present in the gel during electrophoresis in different electric field gradients. When corrected to a common temperature, the electrophoretic mobilities of DNA fragments less than or equal to 1 kilobase pairs (kbp) in size were independent of electric field strength at all field strengths from 0.6 to 4.6 V/cm if the gels contained less than or equal to 1.4% agarose. The mobilities of larger DNA fragments increased approximately linearly with electric field strength. If the agarose concentration was higher than 2%, the mobilities of all DNA fragments increased with increasing electric field strength. The electric field dependence of the mobility was larger in gels cast and run in Tris-borate buffer (TBE) than in gels cast and run in Tris-acetate buffer (TAE), and was more pronounced in gels without ethidium bromide incorporated in the matrix. Ferguson plots were constructed for the various DNA fragments, both with and without extrapolating the temperature-corrected mobilities to zero electric field strength. Linear Ferguson plots were obtained for all fragments less than or equal to 12 kbp in size in agarose gels less than or equal to 1.4% in concentration if the mobilities were first extrapolated to zero electric field strength. Concave upward curvature of the Ferguson plots was observed for DNA fragments greater than or equal to 2 kbp in size at finite electric field strengths. Convex downward curvature of the Ferguson plots was observed for DNA fragments greater than or equal to 1 kbp in size in agarose gels greater than or equal to 2% in concentration. The mobilities of the various DNA fragments, extrapolated to zero agarose concentration and zero electric field strength, decreased with increasing DNA molecular weight; extrapolating to zero molecular weight gave an "intrinsic" DNA mobility of 2.7 x 10(-4) cm2/Vs at 20 degrees C. The pore sizes of LE agarose gels cast and run in TAE and TBE buffers were estimated from the mobility of the DNA fragments.(ABSTRACT TRUNCATED AT 400 WORDS)     

Curved DNA molecules migrate anomalously slowly in polyacrylamide gels even at zero gel concentration

The electrophoretic mobilities of curved and normal DNA molecules of the same size have been measured in polyacrylamide gels containing various acrylamide concentrations and cross-linker ratios. Ferguson plots were constructed to extrapolate the observed mobilities to zero gel concentration. The DNA samples were two 147-bp restriction fragments, called 12A and 12B, obtained from the MspI digestion of plasmid pBR322, and head-to-tail multimers of each fragment. Fragment 12A is stably curved and migrates anomalously slowly in polyacrylamide gels; fragment 12B has the conformation of normal DNA and migrates with normal electrophoretic mobilities. The extrapolated mobilities of the curved fragment 12A and its multimers at zero gel concentration are lower than the extrapolated mobilities of the normal fragment 12B and its multimers. The free solution mobility of the curved fragment 12A, measured by CE, is also lower than that of the normal fragment 12B. The combined results indicate that the extrapolated mobilities observed for curved DNA molecules at zero polyacrylamide gel concentration reflect the intrinsic differences in their free solution mobilities.     

Molecular sieving of lambda phage DNA in polyacrylamide solutions as a function of the molecular weight of the polymer.

Electrophoresis of lambda phage DNA was carried out in solutions at various concentrations of uncrosslinked polyacrylamide of 0.6, 1, 5 and 9 x 10(6) molecular weight (Mw) with narrow Mw distribution. By inspection of mobilities in the various concentration ranges, it appears that mobilities decrease, and retardation increases, with increasing Mw. The relation between electrophoretic retardation and the Mw of the polymer was also interpreted (i) in the manner previously applied to nonlinear Ferguson plots and compatible with the Ogston model; and (ii) empirically, on the basis of the first derivatives of the functions describing the Ferguson plots at the polymer concentrations used. Interpretation (i) shows that the retardation increases linearly in the order of 0.6, 1, 5 and 9 x 10(6) Mw of polyacrylamide. Interpretation (ii) shows a nonlinear increase of retardation in the Mw range 5 to 9 x 10(6), and a decrease in retardation as Mw is raised from 0.6 to 5.0 x 10(6). Hypothetically, interpretation (ii) can be explained mechanistically by a progressive change, as the polymer size is increased, from a collision with the surface of the polymer fiber to one occurring after permeation in the interior of a random-coiled fiber. Interpretation (i) may fail to detect that change due to the large difference between DNA mobility in solutions of the smallest polymer and the free mobility. DNA peak detection in all of the four size classes of polyacrylamide in solution is limited to relatively narrow ranges of polymer concentration.(ABSTRACT TRUNCATED AT 250 WORDS)     

The electrophoretic properties of a DNA cube and its substructure catenanes.

Electrophoretic properties have been measured for a DNA molecule whose helix axes have the connectivity of a cube. This molecule is a topologically bonded complex of six cyclic 80-mer molecules, in which each cycle corresponds to a face of the cube. Each cyclic molecule is doubly catenated to each of its four neighbors. Substructures of this molecule include a 5-cycle structure lacking one strand, two topoisomers of 4-cycle structures and two topoisomers of 3-cycle structures. One 4-cycle structure is a cyclic belt around the cube, lacking a top and a bottom, whereas the other lacks two catenated strands, such as the top and the front. One 3-cycle structure is a linear belt of three cycles, and the other corresponds to the three cycles that surround a corner. Each of these molecules is separable from the others under appropriate gel conditions. We have measured mobilities and calculated Ferguson plots for each of these molecules on polyacrylamide gels under both native and denaturing conditions. The measurements have been made with 1.25, 2.5, and 5% crosslinking of the gels. The data show that the higher-symmetry 3-cycle and 4-cycle structures migrate more slowly than their lower-symmetry isomers, under conditions where their Ferguson plots are parallel.     

Estimation of polyacrylamide gel pore size from Ferguson plots of normal and anomalously migrating DNA fragments. I. Gels containing 3% N,N'-methylenebisacrylamide

The mobilities of normal and anomalously migrating DNA fragments were determined in polyacrylamide gels of different acrylamide concentrations, polymerized with 3% N,N'-methylenebisacrylamide as the crosslinker. The DNA samples were a commercially available 123-bp ladder and two molecular weight ladders containing multiple copies of two 147-base pair (bp) restriction fragments, obtained from the MspI digestion of plasmid pBR322. One of the 147 bp fragments is known to migrate anomalously slowly in polyacrylamide gels. Ferguson plots were constructed for all multimer ladders, using both absolute mobilities and relative mobilities with respect to the smallest DNA molecule in each data set. If the retardation coefficients were calculated from the relative mobilities, and the rms radius of gyration was used as the measure of DNA size, the Ogston equations were obeyed and the gel fiber parameters could be calculated. The effective pore sizes of the gels were estimated from the gel concentration at which the mobility of a given DNA molecule was reduced to one-half its mobility at zero gel concentration. The estimated pore radii ranged from approximately 130 nm for 3.5% gels to approximately 70 nm for 10.5% gels. These values are much larger than the pore sizes previously determined for the polyacrylamide matrix.     

Resolution of 8-aminonaphthalene-1,3,6-trisulfonic acid-labeled glucose oligomers in polyacrylamide gel electrophoresis at low gel concentration

A discontinuous Tris-Cl/acetate (OAc) buffer system, unprecedently containing OAc as the trailing constituent, and operative in polyacrylamide gel electrophoresis (PAGE) at low polyacrylamide concentration (T = 4.8%) is described in the paper. The characteristics of the electrophoretic system are illustrated by the resolution of fluorescent 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS)-labeled malto-oligosaccharides and dextran homopolymers. In this buffer system, the resolving phase is constituted by Tris-OAc behind a moving boundary formed between the leading chloride ion of Tris-HCl gel buffer and the trailing OAc ion provided by a catholyte of NH(4)OAc. In contrast with the results obtained with Tris-CI/glycinate buffer commonly used in electrophoresis, or with Tris-CI/borate, the best resolution of the glucose oligomers containing 1-4 glucose units in Tris-OAc, pH 8.8, ionic strength of 0.08, was obtained at 4.8% polyacrylamide concentration, using 0.5 M NH(4)OAc, pH 9.5 as the catholyte. Under those conditions, the ANTS-glucose oligomers were separated with mobilities decreasing from glucose to maltohexaose. The linear Ferguson plots (log relative mobility, R(f), vs.%T) of the glucose oligomers show that the surface net charge of those oligomers is inversely related to their sizes, given by the slopes, K(R), of the plots. The molecular weight of the oligomers is directly but nonlinearly related to K(R). The novel electrophoretic system illustrated here for separation of short ANTS-saccharides can be potentially applied to the resolution of other biomolecules such as rapidly migrating DNA, peptides or proteins.     

Analysis of one-dimensional gels and two-dimensional Serwer-type gels on the basis of the extended Ogston model using personal computers

This report presents the stand-alone computer application ELPHOFIT, a software package for the analysis of gel electrophoretic data based on Ferguson plots. Either conventional one-dimensional gels or two-dimensional agarose gels (Serwer-type) can be evaluated. Special emphasis is on the latter gel type, which has been applied previously for the separation of DNA, intact viruses and polydisperse meningitis vaccines. ELPHOFIT is designed for Macintosh PCs and for the IBM XT, AT, PS/2 and compatibles. The program operates interactively with the user, who determines the course of evaluation. Data input is in the format of files providing values of gel electrophoretic migration distances or particle mobility (absolute or relative). Data processing involves a simultaneous least-square curve fitting algorithm (Newton-Gauss, Marquardt-Levenberg) which uses equations derived from the extended Ogston model. Functions are fit to the database by adjusting their variables, representing physical parameters of the gel and the electrophoresed particle. The program output consists of tables and graphics accompanied by an explanatory text providing the following information: (i) radius and free mobility of the electrophoresed particle, (ii) fiber radius, length and volume, mean or median pore radius of the gel, (iii) linear Ferguson plots, (iv) iso-free-mobility/iso-size nomogram for two-dimensional gels, (v) confidence ellipses, (vi) required parameters for image processing program GELFIT and (vii) goodness-of-fit and other statistical parameters, such as standard errors, dependency values, root-mean-square (RMS) error and determination coefficient. Other features of the program are (i) simulation of Serwer-type two-dimensional electrophoresis, (ii) standardization according to size, or size and free mobility, (iii) the conversion of particle radii to molecular (or particle) weight and vice versa, (iv) interconversion of DNA size specifications, i.e. the number of base pairs and the geometric mean radii, (v) computation of gel concentration for optimal resolution of two components, (vi) option to obtain a session record, (viii) option to establish a data output file containing the information of generated graphics (IBM only) and (ix) a text editing facility, e.g., for creating data files. Graphics (Macintosh version, PICT format) and text output files (both IBM and Macintosh versions, standard ASCII format) generated by ELPHOFIT are compatible with commercially available software.     

Resolution of a paradox in the electrophoresis of DNA in agarose gels

A paradox was observed in a previous study of the electrophoresis of linear DNA fragments in agarose gels (D. L. Holmes and N. C. Stellwagen, Electrophoresis 1990, 11, 5-15). The pore size of the agarose matrix was more accurately determined if the root-mean-square radius of gyration was used to measure DNA macromolecular size. However, the Ogston equations were obeyed and other gel parameters such as the apparent fiber radius and fiber volume appeared to be better described if the geometric mean radius was used to measure DNA size. This paradox can be resolved if relative mobilities (with respect to the smallest DNA molecule in the data set) are used to construct the Ferguson plots, instead of absolute mobilities. Using relative mobilities and the root-mean-square radius of gyration, the Ogston equations are obeyed and the pore size of the matrix is consistent with values determined by other methods.     

Detection of conformational and net charge differences in DNA-protein complexes by quantitative electrophoresis on polyacrylamide-agarose copolymer gels

The Galactosidase repressor (GalR) of Escherichia coli modulates the expression of the gal operon by binding to two DNA operators, OE and O1. The OE and O1 elements are 16 bp pallindromic DNA sequences, differing in four of the base pairs. OE and O1 DNA fragments, both free and complexed with repressor, were analyzed by "quantitative gel electrophoresis". By the criteria of that method, applied to the linear Ferguson plots of both DNA fragments and the linear ranges of those of the DNA-GalR complexes, it was shown that the apparent size of DNA increases upon repressor binding. Moreover, this size increase is greater for the complex with the O1 operator than for the complex with the OE operator in the case that GalR is located in the center of a 155 bp DNA fragment. This is not the case when GalR is located in a peripheral position. By contrast with their size differences, the centrally located GalR-O1 and GalR-OE complexes appear to possess indistinguishable net surface charge densities as judged from the intercepts with the mobility axis. The larger size of the complex with centrally located O1 fragment, as compared with that bearing the OE fragment, is interpreted as being due to bending of the DNA-protein complex, since an authentically bent fragment of a plasmid with bent upstream activator sequence also exhibits a larger slope of the Ferguson plot, and thus the larger size, than predicted on the basis of its DNA chain length (bp).(ABSTRACT TRUNCATED AT 250 WORDS)     

Gel electrophoretic analysis of DNA branched junctions

Gel electrophoresis has provided much of the detailed information we have about the properties of DNA junctions, stable branched molecules formed from oligonucleotide or polynucleotide strands. Here we review these applications, and present the results of an electrophoretic investigation of conformationally restricted junctions formed by covalently connecting two different pairs of strands in a junction with four arms. Native gel electrophoresis is employed to establish the formation and stoichiometry of the multistrand complexes. Ferguson analysis of native gel mobility shows that junctions have retardation coefficients that are distinct from those of linear DNA duplexes. Denaturing gel electrophoresis is the primary tool for characterizing junctions that have been covalently linked together to form both linear and macrocyclic oligomers of junctions (oligojunctions). Radioactively labelled strands enable one to monitor the progress of the ligation reaction: both linear and closed cyclic molecules result, and these can be distinguished by applying Ferguson analysis to denaturing gels. Combinations of exonuclease III, restriction enzymes and sequencing reactions have been applied to oligojunction molecules, and the results are all analyzed on denaturing gels. Junctions containing intramolecular "tethers" that restrict the conformation freedom of the complex comprise a new system for analyzing the conformations of branched molecules. In these tethered junctions, the ability of arms to move relative to each other is restricted substantially by covalently connecting pairs of arms in the original complex with short, flexible loops. The two tethers used here constrain the helical domains of the structure to be roughly parallel or anti-parallel. In this article, we use Ferguson analysis to compare two tethered junctions with an untethered junction. At high gel concentrations, the mobility of the untethered complex is found to be closer to that of the molecule tethered anti-parallel than to the one tethered parallel. Curvature in the Ferguson plots for all three of these junctions is detected over a range of compositions. At low gel concentrations, differences in electrophoretic mobility persist, suggesting that the untethered junction differs in charge as well as conformational freedom from the tethered analogs. We expect that studies of this kind will be able to define the conformational repertoire of junctions of different kinds, and to explore the effects of electrophoresis on these states.     

Sieving of ionic constituents across moving boundaries in gel electrophoresis

The representative beta-hydroxyethylmorpholinium-chloride-bicinate moving boundary with a trailing ion net mobility relative to Na+ of 0.41, detected by precipitation of chloride with silver nitrate, exhibits a decreasing chloride mobility at increasing polyacrylamide gel concentrations from 3.5 to 45%T, 5%CBis. This decrease, largely due to an increase of field strength at constant current, is described by a convex* plot of log (mobility) vs. %T (Ferguson plot) and signifies that chloride/bicinate are sieved by the gel. In agarose gels, the same plot of mobility vs. gel concentration is constant below 7% gel concentration, since in those gels field strength and migration rate remain the same within that gel concentration range. Both in polyacrylamide and in agarose gels the displacement rate of the chloride-bicinate boundary as a function of the time of electrophoresis or distance migrated remains invariant within 15%. The plot of log (mobility) vs. gel concentration extrapolated to 0%T is 5.85 and 5.41 (10(-5) cm2s-1V-1) for polyacrylamide and for agarose (SeaKem HGT-P,FMC) gels, respectively. The slightly decreased mobility intercept at 0%T for agarose is presumably due either to the electroendosmotic properties of agarose HGT-P and/or failure to Sufficiently take into account the flattening of the Ferguson plot in the polyacrylamide concentration range below 3% in which a transition from a gel to a fluid (sol) medium takes place.     

Information on DNA conformation derived from the Ferguson plot of DNA fragments of up to 9 kb in size, using polyacrylamide gel electrophoresis in a discontinuous buffer system.

The Ferguson plot in polyacrylamide gel electrophoresis (PAGE)(15%CDATD, moving boundary electrophoresis buffer system operative at pH 8.9, 4 degrees C, 8 mA/cm2 of gel) of DNA fragments up to 9.4 kb in size was found to exhibit a linear segment at polyacrylamide concentrations starting at 3% T and undergoing a gradual transition into a concave segment at higher gel concentrations, confirming previous findings by Stellwagen. The larger the DNA, and the higher the gel concentration, the less extended the linear and the more extended the concave segment of the plot. The lowest % T of the linear range for DNA in polyacrylamide remains unknown since mobilities at nongelling concentrations below 3% T have not as yet been measured. As previously suggested, the transition from the linear to the concave segment corresponds to that from the randomly oriented DNA to the anisotropically stretched, "reptating" DNA. For a DNA of 9.4 kb in size, the end of the linear range of the Ferguson plot can be extended from 3.5 to 5% T when 15% DATD rather than 2.5% Bis is used to crosslink the polyacrylamide. Increasing the temperature of PAGE from 4 degrees C to 25 and 50 degrees C widens the linear segment progressively, indicating an increasingly random orientation with rising temperature. When current density is increased from 8 to 40 mA/cm2, the concave curvature of the Ferguson plot of DNA 1 to 9.4 kb in size decreases, suggesting a transition from a "reptating" to a randomly distributed molecule, due to increased Joule heat.(ABSTRACT TRUNCATED AT 250 WORDS)     

Procedures and computer program for deriving the Ferguson plot from electrophoresis in a single pore gradient gel: application to agarose gel and a polystyrene particle.

This study presents a computerized evaluation of pore gradient gel electrophoretograms to arrive at estimates for both the particle-free mobility and retardation coefficient, which is related to particle size. Agarose pore gradient gels ranging from 0.2 to 1.1% agarose were formed. Gel gradients were stabilized during their formation by a density gradient of 0-20% 5-(N-2,3-dihydroxypropylacetamido)- 2,4,6-triiodo-N,N'bis-(2,3-dihydroxypropyl)-isophthalamide (Nycodenz). Densitometry of gelled-in Bromophenol Blue showed that these pore gradients exhibited a linear central segment and were reproducible. Migration distances of polystyrene sulfate microspheres (36.5 nm radius) in agarose pore gradient gel electrophoresis were determined by time-lapse photography at several durations of electrophoresis. These migration distances were evaluated as a function of migration time as previously reported (D. Tietz, Adv. Electrophoresis 1988, 2, 109-169). Although this is not necessarily required, the mathematical approach used in this study assumed linearity of both the pore gradient and the Ferguson plot for reasons of simplicity. The data evaluation on the basis of the extended Ogston model is incorporated in a user-friendly program, GRADFIT, which is designed for personal computers (Macintosh). The results obtained are compared with (1) conventional electrophoresis using several gels of single concentration with and without Nycodenz, and (ii) a different mathematical approach for the analysis of gradient gels (Rodbard et al., Anal. Biochem. 1971, 40, 135-157). Moreover, a simple procedure for evaluating linear pore gradient gels using linear regression analysis is presented. It is concluded that the values of particle-free mobility and retardation coefficient derived from pore gradient gel electrophoresis using the different mathematical methods are statistically indistinguishable from each other. However, these values are different, albeit close, to those obtained from conventional Ferguson plots. One of the possible reasons for this relatively minor discrepancy is that the particle-free mobility changed slightly during electrophoresis, which has a different effect on electrophoresis in homogeneous gels (single time measurement) and pore gradient gels (multiple time measurements). The characterization of particles according to size and charge by pore gradient electrophoresis provides a significant operational simplification and sample economy compared to that requiring the use of several gel concentrations, although at the price of increased requirements of instrumentation.     

Suppression of electroosmotic flow and its application to determination of electrophoretic mobilities in a poly(vinylpyrrolidone)-coated capillary

A hydrophilic polymer, poly(vinylpyrrolidone) (PVP), was employed for suppressing the electroosmotic flow (EOF). A capillary was filled with aqueous PVP solution for coating the capillary wall with PVP; the PVP solution was then replaced by a migration buffer solution containing no PVP. Three types of PVP with different molecular weights were examined. The EOF was suppressed more effectively as the molecular weight of PVP increased. The EOF in the coated capillary was approximately 10-fold smaller than that of a bare capillary and was constant in the pH range of 6-8. The suppressed EOF was stable even when no PVP was added to the migration buffer. However, the EOF increased significantly when sodium dodecyl sulfate was added into the migration buffer. The method was applied for determining the electrophoretic mobilities of inorganic anions that have negative electrophoretic mobilities larger than the electroosmotic mobility of the bare capillary. A novel method for determining the electrophoretic mobilities was proposed based on the linear relationship between electric current and electrophoretic mobility. The electrophoretic mobility was proportional to the electric current. Therefore, the intercept of the regression equation represents the electrophoretic mobility at room temperature. The electrophoretic mobilities were in good agreement with the absolute electrophoretic mobilities.     

Information on DNA conformation derived from transverse pore gradient gel electrophoresis in conjunction with an advanced data analysis applied to capillary electrophoresis in polymer media.

Abnormally slow migration of DNA is conventionally viewed as being due to an abnormal conformation relative to "linear" standards. The evidence for this rests on a few instances where nonlinear DNA structures have been established by independent methods and yield low mobilities relative to standards. Transverse pore gradient gel electrophoresis of authentically bent kinetoplast DNA and of an upstream activator sequence (UAS) of an E. coli operon promoter shows in addition that curves of migration distance vs. gel concentration ("Ferguson curves") of such abnormally conformed DNA differ from those of "linear" standards. Since Ferguson curves are interpretable with regard to molecular size in concordance with a mathematical model (Ogston model), transverse pore gradient gel electrophoresis provides a simple means of correlating abnormally slow migration of DNA with molecular size. In addition, transverse pore gradient gel electrophoresis is able to distinguish between DNA banding which exhibits a steeper dependence on gel concentration than "linear" standards from one which shows the same dependence. The former appears characteristic of circularly bent DNA and gives rise to a substantial retardation, the latter of bending across a knot or kink in the DNA chain associated with a relatively minor retardation relative to standards. Circularly bent restriction fragments formed from kinetoplast DNA retain the characteristic intersecting Ferguson curves on the transverse pore gradient gel. Another authentically "abnormal" DNA structure recognizable on transverse pore gradient gels is supercoiled DNA derived from the reaction of topoisomerase with a plasmid. Different lengths of supercoiled sequences give rise to parallel Ferguson curves clearly intersecting with those of linear standards.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

SDS-CGE of proteins in microchannels made of SU-8 films

This work describes the SDS-CGE of proteins carried out in microchannels made of the negative photoresist EPON SU-8. Embedded electrophoretic microchannels have been fabricated with a multilayer technology based on bonding and releasing steps of stacked SU-8 films. This technology allows the monolithic integration of the electrodes in the device. A high wafer fabrication yield and mass production compatibility guarantees low costs and high reliability. A poly(methyl methacrylate) (PMMA) packaging allows an easy setup and replacement of the device for electrophoresis experiments. In addition, the wire-bonding step is avoided. The electrophoretic mobilities of four proteins have been measured in microchannels filled with polyacrylamide. Different pore sizes have been tested obtaining their Ferguson plots. Finally, a separation of two proteins (20 and 36 kDa) has been carried out confirming that this novel device is suitable for protein separation. A resolution of 2.75 is obtained. This is the first time that this SU-8 microfluidic technology has been validated for SDS-CGE of proteins. This technology offers better separation performance than glass channels, at lower costs and with an easy packaging procedure.