Early melting of supercoiled DNA.

Denaturing gradient gel electrophoresis (formamide with urea) has been used to study the melting of supercoiled DNA. A linear gradient of denaturant concentration proportional to a 25 degrees C linear increase of temperature (Teff) from the left to the right edge of the gel was created perpendicular to DNA migration. The mobility of supercoiled DNA molecules was shown to drop to the level of relaxed molecules a long way (5-30 degrees C) before linear DNA began to melt. The further increase of Teff, including the melting range for linear molecules, caused no appreciable changes in the mobility of relaxed molecules. The transition curves are S-shaped for all the topoisomers, and an increase of superhelicity shifts the transition towards lower Teff values. The analysis of the results indicates that the observed relaxation of superhelical molecules is due to denatured region forming in them, their size increasing with the topoisomer number.     

Electrophoretic separation of S. pombe chromosomes in polyacrylamide solutions using a constant field

Previous electrophoretic separations of megabase (Mb) sized DNA have been achieved in pulsed electric fields, using agarose gel as a matrix. The present study demonstrates separations of Mb sized DNA due to a retardation of migration in proportion to the concentration of uncrosslinked polyacrylamide of 5 x 10(6) molecular weight, using a constant electric field. Potentially, the method should be applicable to large DNA in general, greatly reducing the instrumental complexity of such separations and rendering them compatible with capillary electrophoresis apparatus.     

Transverse agarose pore gradient gel electrophoresis of DNA.

Transverse agarose pore gradient gels were prepared on GelBond in the concentration range of nominally 0.2-1.5% SeaKem GTG agarose, using density stabilization by glycerol and incorporation of a dye to define the gel concentration at each point on the pore gradient gel. The distribution of the dye was evaluated by photography, video-acquisition and digitization of the gradient mixture and by densitometry of the gel. The gel was applied to the electrophoresis of a 1-kb standard ladder of DNA fragments, using standard submarine apparatus. The method extends to agarose gel electrophoresis the benefits of semi-automated analysis of 'Ferguson curves' described in application to polyacrylamide gel by Wheeler et al. (J. Biochem. Biophys. Methods 24, 171-180).     

Estimation of polymerization efficiency in the formation of polyacrylamide gel,using continuous optical scanning during polymerization

The Ferguson plot and ‘quantitative’ gel electrophoresis (based on the Ferguson plot) depend on a knowledge of accurate gel concentrations. The easiest way to estimate accuracy of gel concentrations, in terms of the degree of completion of the polymerization reaction which gives rise to a gel, is by spectrophotometry. Making use of the apparatus for continuous optical scanning of polyacrylamide gels, the extent and rate of polymerization of cross-linked polyacrylamide were estimated by measuring the absorbance at 275 mm of the reaction mixture subsequent to free radical initiation of polymerization. Under appropriate conditions of monomer concentration, initiator levels and temperature, absorbance decreased monotonically after alag period of 10 min, and after 20–30 min of reaction the absorbance reached a plateau value which provided a measure of polymerization efficiency. Application of a standard curve of absorbance vs. monomer concentration allowed one to quantitate concentrations of residual monomer throughout the course of polymerization. Under a set of arbitrary polymerization conditions (e.g. 6–20% total gel concentration), the reaction went to 63–96% completion. The rate of polymerization was approximately proportional to the square of the monomer concentration (2nd-order reaction kinetics). Absorbance decrease subsequent to the initiation of the polymerization reaction appeared suitable as a measure of efficiency of polymerization since: (1) absorbance spectra of monomers at 0.5%T and residual monomers in a 10%T gel, at a time when polymerization seemed terminated, coincided; (b) values of residual monomer obtained were reasonable (10–30%); (c) bimolecular reaction kinetics were found, in agreement with expectation; and (c) absorbance of incomplete polymerization mixtures, deficient in either initiators or monomers, was constant with time.     

Rapid determination of protein molecular weight by the Ferguson method and multiplexed capillary electrophoresis

A method based on the use of the Ferguson method and multiplexed sodium dodecyl sulfate-capillary gel electrophoresis (SDS-CGE) with UV detection was demonstrated for the rapid determination of molecular weights of proteins. The method employs a capillary array where different uncoated capillaries are filled with gel buffers containing different concentrations of poly(ethylene oxide) (PEO). All data required to construct Ferguson plots and universal calibration curves for the determination of the molecular weights of diverse types of proteins are generated simultaneously in an eight-capillary array within 20 min.     

The thrombin binding aptamer GGTTGGTGTGGTTGG forms a bimolecular guanine tetraplex

In the literature, the thrombin binding aptamer GGTTGGTGTGGTTGG is generally taken as a prototype of an intramolecular guanine tetraplex of DNA. Our results, however, show that this notion is not true in aqueous solutions. This conclusion is based on a dependence of the CD spectra on aptamer concentration, migration of the aptamer in polyacrylamide gels, and the Ferguson analysis of the gel migration data. The presented data document that the aptamer forms a bimolecular tetraplex. We furthermore show that only an extension of the aptamer by a sequence containing further guanines, or an elongation of loop regions, causes that its tetraplex folding is intramolecular.     

Reevaluation of the electrophoretic migration behavior of soluble globular proteins in the native and detergent-denatured states in polyacrylamide gels

Although sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis is widely used for estimating molecular masses of proteins, considerable uncertainty still exists both about the structure of SDS-protein complexes and about their mechanism of electrophoretic migration. In this study, soluble globular proteins, with masses of 14-200 kDa, were heat-denatured in the presence of SDS and their relative total molecular volume and net charge were estimated from Ferguson plots of electrophoretic mobility vs acrylamide concentration. Native globular protein served as standards for overall molecular size and effective radii. Results revealed at least two independent electrophoretic migration mechanisms for the SDS-protein complexes: (i) for proteins in the 14-65 kDa range at <15% acrylamide, linear Ferguson plots suggested that they migrated ideally and that their effective radii could be estimated in this manner: (ii) concave plots at higher gel concentrations, and for complexes derived from larger proteins, indicated that migration in these cases could be described by reptation theory. Migration of the large proteins at lower gel concentrations and small proteins at higher gel concentrations was not well described by either theory, representing intermediate behavior not described by these mechanisms. These data support models in which all but the smallest SDS-protein complexes adopt a necklace-like structure in which spherical micelles are distributed along the unfolded polypeptide chain. Possible relations to recent alternative models of gel electrophoresis are also discussed.     

Topological effects on the electrophoretic mobility of rigid rodlike DNA in polyacrylamide gels

The electrophoretic migration of rigid rodlike DNA structures with well defined topologies has been investigated in polyacrylamide (PA) hydrogels prepared by copolymerization of acrylamide and N, N'-methylenebisacrylamide. Previous studies have reported structural and dynamic characteristics of linear and branched DNA during electrophoresis in PA gels using a variety of experimental parameters. However, a thorough investigation aimed at establishing specific relationships between topological features of rigid rodlike DNA structures and their electrophoretic behavior is still needed. In order to study these topological effects on mobility, an intensive examination of the electrophoretic mobility of small linear and starlike DNA was performed. A series of model DNA structures with well-defined branched topologies were synthesized with varying molecular parameters, such as number of arms surrounding the branch point and arm length. The electrophoretic mobility of these structures was then contrasted with a series of data obtained using linear DNA of comparable molecular size. When large DNA stars (M >/= 60 bp) were compared with linear DNA of identical molecular weight, the Ferguson plots were quite different. However, small DNA stars (24-32 bp) and linear analogues had identical Ferguson plots. This indicates that a different motional mode or greater interaction with the gel exists for the larger DNA stars. When the total molecular weight of the DNA stars was held constant and the number of arms varied, the Ferguson plots for all the stars were identical. Additionally, a critical pore size was reached when the ratio of linear DNA mobility to star DNA mobility increased dramatically. Thus, while the incorporation of a single branch point can produce a large reduction in mobility, above a critical molecular size, the incorporation of additional branch points does not appear to provide further reduction in mobility. This finding is consistent with the transport properties of large synthetic star polymers, where a large reduction in their diffusion coefficient is observed when a single branch is added. When additional arms are incorporated, large synthetic stars do not display an appreciable further reduction in diffusion coefficient. The effect of arm length on mobility for rigid rod DNA stars was also studied. For four-arm DNA stars, the mobility was found to scale as an exponential function of the arm length. Finally, a recently proposed phenomenological model was used to successfully fit the mobility data for linear rigid rod DNA at various concentrations of PA.     

pK determinations via pH-mobility curves obtained by isoelectric focusing-electrophoresis: Theory and experimental verification

Basic equations have been derived linking the electrophoretic migration in a stationary pH gradient of simple, singly charged cations or anions and of mono- mono- valent ampholytes with the pKs of their ionizable groups. In the case of diprotic ampholytes, an equation and a curve are described calculating a correction factor to be applied to the mobility measurements, accounting for the influence of the opposite charge species on the mobility curve of the ion being measured. This correction factor is a function of ΔpK and increases exponentially with decreasing values of ΔpK. These theoretical considerations have been experimentally verified by running pH-mobility curves of colored compounds, such as methyl red, neutral red and dexorubicin. The pKs thus measured were in excellent agreement with the pKs obtained independently by spectrophotometric titrations.     

Parameters of field inversion gel electrophoresis for the analysis of pox virus genomes.

The effects of variation in the lengths of forward and reverse pulses, voltage gradient, gel concentration and gel temperature on the mobility of DNA molecules in agarose gels during field inversion gel electrophoresis (FIGE) have been determined. A curve, which best fits the empirical data, is presented and allows the choice of pulse conditions and voltage gradient most suitable for the resolution of molecules of chosen size. The use of FIGE in the analysis and direct mapping of large virus genomes is illustrated using vaccinia virus DNA.     

The sodium channel from rat brain. Separation and characterization of subunits

Procedures are described for separation of the alpha, beta 1, and beta 2 subunits of the voltage-sensitive sodium channel from rat brain by gel filtration in sodium dodecyl sulfate (SDS) before and after reduction of intersubunit disulfide bonds or by preparative SDS-gel electrophoresis. Partial proteolytic maps of the SDS-denatured subunits indicate that they are nonidentical polypeptides. They are all heavily glycosylated and contain complex carbohydrate chains that bind wheat germ agglutinin. The apparent molecular weights of the separated subunits were estimated by gradient SDS-gel electrophoresis, by Ferguson analysis of migration in SDS gels of fixed acrylamide concentration, or by gel filtration in SDS or guanidine hydrochloride. For the alpha subunit, SDS-gel electrophoresis under various conditions gives an average Mr of 260,000. Gel filtration methods give anomalously low values. Removal of carbohydrate by sequential treatment with neuraminidase and endoglycosidase F results in a sharp protein band with apparent Mr = 220,000, suggesting that 15% of the mass of the native alpha subunit is carbohydrate. Electrophoretic and gel filtration methods yield consistent molecular weight estimates for the beta subunits. The average values are: beta 1, Mr = 36,000, and beta 2, Mr = 33,000. Deglycosylation by treatment with endoglycosidase F, trifluoromethanesulfonic acid, or HF yields sharp protein bands with apparent Mr = 23,000 and 21,000 for the beta 1 and beta 2 subunits, respectively, suggesting that 36% of the mass of the native beta 1 and beta 2 subunits is carbohydrate.     

Analysis of genetic mutations in human lactate dehydrogenase-A(M) deficiency using DNA conformation polymorphism in combination with polyacrylamide gradient gel and silver staining

Human lactate dehydrogenase (LDH)-A mutant gene was analyzed by polymerase chain reaction - DNA conformation polymorphism (DCP). We used polyacrylamide gradient gel and silver staining procedures for DCP analysis and observed abnormal migration patterns in individuals heterozygous for LDH-A deficiency. Further sequence determination of the mutant alleles consistently resulted in detection of base substitutions, a G to T transversion at codon 328 (GAG----TAG), and synonymous substitutions at codon 115, 160 and 172. Such mutations were easily detectable using the DCP technique. The DCP technique using the polyacrylamide gradient gel and silver staining method seems likely to be useful for the rapid screening of mutations and for further genotype detection.     

Glass micromodel study of bacterial dispersion in spatially periodic porous networks

Successful implementation of bioremediation clean-up strategies depends on accurate predictions of the transport of bacteria within the subsurface. In this study, etched flat-plate glass micromodels were used to examine bacterial transport in a homogenous network. These networks were created by acid-etching interconnected channels into a glass plate and then fusing it to an unetched plate forming semi-cylindrical pores. The transparent nature of the micromodel allows for both qualitative observations of the bacteria within the pores and quantitative measurements of their concentration. The micromodels are designed to allow establishment of a well-characterized step change in bacterial concentration (Escherichia coli NR50) within the network. During the experiments, bacteria are dispersed through the network by flow. Light scattering is used to detect the change in turbidity within the pores as the bacteria travel through the network. The change in turbidity is used to construct breakthrough curves and spatial concentration profiles of bacteria within the network. The breakthrough curves are fit to the one-dimensional advection/dispersion equation to determine dispersion coefficients at different interstitial fluid velocities. From the breakthrough curves, dispersion coefficients were reproducible for replicate experiments over a range of velocities in the advection-dominated regime. The dispersivity values for two network designs resembling an interconnecting capillary network and a spatially periodic network of cylinders were 0.28 and 0.33 cm respectively, which are slightly greater than the literature values found for other pore networks. Experiments were also conducted within the diffusion-dominated regime to examine the effects of bacterial motility on dispersion. The accumulation of bacteria on the pore walls became significant at the low flow rates and extended experimental times thereby rendering the use of light scattering to determine concentrations ineffective. Bacterial chemotaxis, created by a self-imposed oxygen gradient, was also observed in the micromodel under stagnant fluid conditions.     

Capillary electrophoresis as a technique to analyze sequence-induced anomalously migrating DNA fragments.

Sequence-induced anomalous migration of double-stranded (ds) DNA in native gel electrophoresis is a well known phenomenon. The retardation of migration is more obvious in polyacrylamide compared with agarose gels, and is greatly affected by the concentration of the gel and the temperature. This anomalous migration results in a difference between calculated and actual sizes of the affected DNA fragments. A low viscosity polymer solution (DNA Fragment Analysis Reagent) under investigation for use in dsDNA analysis by capillary electrophoresis is shown to be useful for the visualization of anomalies in migration of dsDNA fragments. Comparable with traditional slab gel systems, the retardation effect, indicative of bent or curved DNA, is strongly dependent on polymer concentration and separation temperature. These dependencies have implications on the accurate sizing of dsDNA fragments with unknown sequences and secondary structures.     

Orientation of DNA molecules in agarose gels by pulsed electric fields

The electric birefringence of DNA restriction fragments of three different sizes, 622, 1426, and 2936 base pairs, imbedded in agarose gels of different concentrations, was measured. The birefringence relaxation times observed in the gels are equal to the values observed in free solution, if the median pore diameter of the gel is larger than the effective hydrodynamic length of the DNA molecule in solution. However, if the median pore diameter is smaller than the apparent hydrodynamic length, the birefringence relaxation times increase markedly, becoming equal to the values expected for the birefringence relaxation of fully stretched DNA molecules. This apparent elongation indicates that end-on migration, or reptation is a likely mechanism for the electrophoresis of large DNA molecules in agarose gels. The relaxation times of the stretched DNA molecules scale with molecular weight (or contour length) as N2.8, in reasonable agreement with reptation theories.     

A study of electrophoretic mobility of DNA in agarose and polyacrylamide gels

The aim of this paper is to clarify the mechanism of gel electrophoresis of DNA under constant-field conditions. We have conducted a large number of experiments on double-stranded DNA varying in length between approximately 10 and approximately 50,000 base-pairs, in both agarose and polyacrylamide gels ranging from 0.5% to 12% concentration, and with electric field strengths ranging from 0.5 to 8 V/cm. We have made (logarithmic) plots of velocity against length of DNA for all of the various test conditions. At the left-hand side of these plots, all of the empirical curves have a unique, standard shape. When the curves are normalized so that their left-hand parts coincide, a second feature emerges in that, while for any given test the curve follows the "master curve" up to a certain point, it then "breaks away" and becomes horizontal. We describe these two patterns of behaviour as "regions 1 and 2", respectively. We find simple yet comprehensive empirical formulae that fit the observations in the two regions of behaviour: these express the velocity in terms of length of DNA, electric field strength and gel concentration. We then construct two separate theories for the two regions of behaviour. The first theory involves the statistics of motion of an object through a random array of gel obstacles, with the instantaneous speed depending on the number of obstacles with which the object is currently in contact. The second theory is based on the mechanical hypothesis (for which there is other, independent support) that the DNA moves through the gel by piling up against a barrier, which eventually breaks or deforms under the resulting force, thereby allowing the DNA to move on to the next barrier. The statistical theory is an adaptation of existing work, while the mechanical one is new. We also describe experiments on the migration of repeated-sequence, curved DNA with length up to 1500 base-pairs, and we discuss its behaviour in terms of our two theories. Our studies by electron microscopy are consistent with the view that this repeated-sequence DNA adopts a superhelical configuration. Finally, we show that a very wide range of observations may be understood clearly by means of our two theoretical schemes.     

FEULGEN HYDROLYSIS OF NORMAL CELLS AND MOUSE ASCITES TUMOR CELLS

The effect of HCl hydrolysis on the dye content (Feulgen reaction) of normal cells and mouse ascites tumor cells was examined by means of cytophotometric measurements. After 11 min of hydrolysis, 16-day-old tumor cells showed a hypotetraploid DNA line with doubling peaks. The DNA values were in the ratios of 1:2:4:8 during all the tested hydrolysis times (3 to 21 min). The size of the nucleus and the DNA concentration did not influence the hydrolysis and the dye content. However, the time of the hydrolysis considerably influenced the dye content of normal and tumor cells. The course of the curves obtained by plotting dye absorption against hydrolysis time showed an inflection of the curve at 9 min' hydrolysis time in tumor cells, whereas the inflection occurred at 8 min in mitotic cells. These inflections were statistically significant. The DNA stem-line¹ for tumor cells shifted during different hydrolysis times when compared to normal cells. The possibility is discussed of two types of DNA which differed in their acid sensitivity and which yielded atypical hydrolysis curves.     

Structural and enzymological characterization of the homogeneous deoxyribonucleic acid polymerase from Mycoplasma orale.

We have purified the DNA polymerase from Mycoplasma orale to homogeneity. The protein structure of the enzyme was declined by sodium dodecyl sulfate gel electrophoresis, which revealed a single band of 116 000 daltons that was coincident with the polymerase activity profile in the final step of DNA--cellulose chromatography, and by two-dimensional gel analysis, which demonstrated a single protein species at pI = 6.8 that was congruent with enzyme activity and contained the same 116 000 polypeptide. although severe enzyme aggregation occurs during nondenaturing gel electrophoresis, a monomer species can be resolved with a Mr of 140 000 by the Ferguson plot analysis. Gel filtration and velocity gradient centrifugation yield a Stokes radius of 4.8 nm and a sedimentation coefficient of 5.6 S, respectively, from which Mr values of 106 000--128 000 can be computed. The different size values suggest that the polymerase molecule is asymmetric. The purified enzyme has a specific activity of approximately 6 x 10(5) units/mg of protein and in completely devoid of exodeoxyribonuclease and endodeoxyribonuclease activities, at exclusion limits of 10(-4)--10(-6%) of the polymerase activity. The mechanism of polymerization is moderately processive, with an average of 14 +/- 4 nucleotides incorporated per binding event, and the "effective template length" on activated DNA is approximately 40 nucleotides.     

Electric birefringence imaging of electrokinetic agarose orientation.

Time-resolved and steady-state electric birefringence imaging with a slow-scan video camera is used to study orientation during DNA agarose gel electrophoresis. The hydrodynamically induced gel distortion is shown to be the major source of birefringence under electrophoresis running conditions and to generate a birefringence image that approximates the image of the DNA concentration gradient in the electric field direction. A fluid kinematic model is presented to describe the spatial distribution of steady-state birefringence and is verified with fluorescence measurements of DNA distribution. The stress-optic coefficient of 1% agarose gel is measured by mechanical compression and used to evaluate the magnitude of the induced strain on the gel during electrophoresis.     

Orientation of DNA Molecules in Agarose Gels By Pulsed Electric Fields

Abstract

The electric birefringence of DNA restriction fragments of three different sizes, 622,1426, and 2936 base pairs, imbedded in agarose gels of different concentrations, was measured. The birefringence relaxation times observed in the gels are equal to the values observed in free solution, if the median pore diameter of the gel is larger than the effective hydrodynamic length of the DNA molecule in solution. However, if the median pore diameter is smaller than the apparent hydrodynamic length, the birefringence relaxation times increase markedly, becoming equal to the values expected for the birefringence relaxation of fully stretched DNA molecules. This apparent elongation indicates that end-on migration, or reptation is a likely mechanism for the electrophoresis of large DNA molecules in agarose gels. The relaxation times of the stretched DNA molecules scale with molecular weight (or contour length) as N^2.8, in reasonable agreement with reptation theories.