Pulsed field electrophoresis

Pulsed field electrophoresis, or PFE, provides good separation between large molecules that under constant field electrophoresis are hard to isolate. This is due to the weak dependence of the constant field mobility on the molecular weight for these large molecules. If a spectrum of relaxation times exists that describes the recovery of the mobility to its constant field value after a reversal of the field, then we show that molecules with differing molecular weights are separated into two groups. Those with short relaxation times are unaffected by the cycling of the field and those with long relaxation times exhibit reduced mobilities. If the molecules adopt conformations that decrease their mobility initially, after a field reversal we demonstrate that a minimum develops in the mobility as a junction of the relaxation time. Using the model we demonstrate that effects of varying the switching times as a function of time. We predict that exponential rather than linear dependencies of the switching times on time increase the range of molecular weights over which enhanced separation can occur.     

Pulsed field gel electrophoresis techniques for separating 1- to 50-kilobase DNA fragments

Conventional agarose gel electrophoresis separates DNA using a static electric field. The maximum size limit for separation of DNA by this method is about 20 kilobase pairs (kb). A number of new electrophoretic techniques which employ periodic reorientation of electric fields permit separation of DNA well beyond this size limit. We sought to determine whether the use of very fast (millisecond) field switching could improve separation of DNA in the size range of 1 to 50 kb. Additionally, we have compared the resolution obtained with each of the different field switching regimens for DNA in this size range. Switching intervals of from 0.2 to 900 ms were used with unidirectional pulsing of a single electric field, with pulsed field gels, and with field inversion gel electrophoresis. Plotting the mobility of DNA as a function of size demonstrates that under the conditions used, each of these techniques offers comparable resolution. We also have examined the separation obtained when field inversion gels are run with forward and reverse fields of equal voltage and different durations, versus using fields of equal duration and different voltages. Field inversion which uses forward and reverse fields of different voltages yields resolution which is superior to the other methods examined.     

Computer-controlled discontinuous rotating gel electrophoresis for separation of very large DNA molecules

The newly designed equipment for alternating field gel electrophoresis which permits the separation of very large DNA molecules and the simultaneous analysis of up to 35 samples is described. The field alternation is effected by intermittently rotating the submerged agarose gel by optitional angles. The time intervals between changes of position are controlled by a computer program driving a simple switching device which was designed to suit any technique using periodic switching or inversion of the electrical field. Because the electrophoresis unit provides an absolutely homogeneous electrical field, no distortion of migration lanes occurs and the resolution is very good. Moreover, by using a switching time interval gradient an almost perfect linear relationship between migration distances and molecule sizes in the range of about 100-1250 kilobase pairs is observed. In two-dimensional separations, different switching time programs for the first and second dimension allow maximum resolution of selected size ranges. Field inversion gel electrophoresis is possible as well. The performance of the method is demonstrated by comparing the chromosome sizes of different yeast strains.     

Separation of chromosomal DNA molecules from yeast by orthogonal-field-alternation gel electrophoresis

A simple agarose-gel apparatus has been developed that allows the separation of DNA molecules in the size range from 50 kb to well over 750 kb, the largest size for which size standards were available. The apparatus is based on the recent discovery that large DNA molecules are readily fractionated on agarose gels if they are alternately subjected to two approximately orthogonal electric fields. The switching time, which was on the order of 20-50 sec in our experiments, can be adjusted to optimize fractionation in a given size range. The resolution of the technique is sufficient to allow the fractionation of a sample of self-ligated lambda DNA into a ladder of approximately 15 bands, spaced at 50 kb intervals. We have applied the technique to the fractionation of yeast DNA into 11 distinct bands, several of which have been shown by DNA-DNA hybridization to hybridize uniquely to different chromosome-specific hybridization probes. In this paper, we describe the design of the apparatus, the electrophoretic protocol, and the sample-handling procedures that we have employed.     

Resolution of DNA molecules greater than 5 megabases by contour-clamped homogeneous electric fields.

Excellent resolution of chromosomal DNA molecules from Saccharomyces cerevisiae, Candida albicans and Schizosaccharomyces pombe has been obtained using alternating contour-clamped homogeneous electric field (CHEF) gel electrophoresis. The largest of these molecules is greater than 5 Mb in size and is resolved after 130 hours in a 0.6% agarose gel at a field strength of 1.3 V/cm and a switching interval of 1 hour. Separation of concatamers of phage lambda DNA reveals four regions of resolution in alternating CHEF gel electrophoresis. There are two regions of good resolution in which mobility approximates a linear function of molecular weight. These are separated by a region of lower resolution and bounded at high molecular weights by a region of little or no resolution. The four regions are of practical and possibly theoretical importance.     

Uptake of fluorescence-labeled dextrans by 10T 1/2 fibroblasts following permeation by rectangular and exponential-decay electric field pulses

The uptake of fluorescence-labeled dextrans by adherent 10T 1/2 murine fibroblasts following electric field pulse application was used as a criterion for the efficiency of electropermeation. The cells in monolayers were permeated by immersing a coaxial electrode in culture dishes. The percentage of cells which exhibited fluorescence uptake following electric field pulse application was measured at independently varying pulse field strength and pulse length. Dextrans with molecular weights equal to or higher than 41,000 dalton require higher field strength or longer pulse time to penetrate the cells. There is no detectable advantage of using a rectangular pulse against using an exponential decay pulse of similar power. The uptake was proportional to the product of the pulse amplitude and duration over the experimental range of 40-950 microseconds and 0.1-14.5 kV/cm. Cell survival decreases at the upper end of this range. The result provides a direct comparison of electric parameters which so far have not been standardized with regard to cell electropermeation.     

The use of poly(2-acrylamido-2-methyl-1-propanesulfonic acid) polymers as spacers for isotachophoresis in sieving gel matrices

The electric field strength gradients generated in isotachophoresis (ITP) may be used for the separation of biomolecules. Poly(2-acrylamido-2-methyl-1-propanesulfonic acid) (polyAMPS) polymers of a uniform distribution of molecular mass were synthesized and used as novel spacers in ITP. Since these polymeric spacers are strongly acidic species, their ionic charges remain constant over a wide pH range, so that their ionic mobilities are governed solely by their molecular masses and not by the pH of the milieu. A modification of ITP known as telescope electrophoresis was used to separate a number of acidic dyes of varying ionic mobility, using polyAMPS polymers as spacers. The resolution obtained was superior to that obtained by polyacrylamide gel electrophoresis (PAGE), due to the focusing effect of the electric field strength gradient. Since these novel polymeric spacers are designed to operate within sieving medium, it was decided to test their suitability for the separation of DNA molecules. DNA molecules up to 1000 bp long were successfully resolved, with a similar resolution to that obtained with conventional PAGE.     

Growth and metabolism of rodents exposed to 60-Hz electric fields

There have been a number of reports in the literature concerning growth-related changes in various animal species exposed to high-strength electric fields. Many of the laboratories reporting such effects have not documented and controlled for the secondary factors that are associated with generating high-strength electric fields (ie, corona, ozone, harmonic distortion, cage vibration, spark discharge). We have designed an exposure system in which we eliminated or minimized these secondary factors, therefore enabling us to examine only the effects of electric fields per se. Sprague-Dawley rats and Swiss-Webster mice were exposed to 60-Hz electric fields at kV/m for up to four months. In 17 individual experiments, we found a greater number of experiments in which the exposed rats had lower body weights than controls. This trend was not evident in data obtained from 14 individual mouse experiments. In more exhaustive growth studies, we found no significant differences in body weights, organ weights, or O₂ consumption between exposed and sham-exposed controls. Our failure to detect any major changes in growth was probably the result of eliminating or minimizing the secondary factors associated with electric field exposure.     

Synchronization of neuron population subject to steady DC electric field induced by magnetic stimulation

Electric fields, which are ubiquitous in the context of neurons, are induced either by external electromagnetic fields or by endogenous electric activities. Clinical evidences point out that magnetic stimulation can induce an electric field that modulates rhythmic activity of special brain tissue, which are associated with most brain functions, including normal and pathological physiological mechanisms. Recently, the studies about the relationship between clinical treatment for psychiatric disorders and magnetic stimulation have been investigated extensively. However, further development of these techniques is limited due to the lack of understanding of the underlying mechanisms supporting the interaction between the electric field induced by magnetic stimulus and brain tissue. In this paper, the effects of steady DC electric field induced by magnetic stimulation on the coherence of an interneuronal network are investigated. Different behaviors have been observed in the network with different topologies (i.e., random and small-world network, modular network). It is found that the coherence displays a peak or a plateau when the induced electric field varies between the parameter range we defined. The coherence of the neuronal systems depends extensively on the network structure and parameters. All these parameters play a key role in determining the range for the induced electric field to synchronize network activities. The presented results could have important implications for the scientific theoretical studies regarding the effects of magnetic stimulation on human brain.     

Electro-optical studies on synthetic polyelectrolytes: IV. Electric polarizability and conformation of poly-N-methyl-2-vinylpyridinium bromide in aqueous solution

Measurements of the relaxation time on aqueous solutions of the title polyelectrolyte as a function of the concentration and of the molecular weight show that its conformation at very high dilution can be better accounted for by a weakly bending rod or worndike chain model, with persistence length ranging from 200 to 400 Å. The analysis of the field strength dependence of the electric birefringence yields electric polarizability values which increase sharply with the dilution and are not significantly dependent upon the molecular weight. This has been tentatively interpreted on the basis of the theories derived by Oosawa and by Mendel and Van der Touw. The partially flexible model proposed by the latter authors allow to identify the electric polarizability calculated from electro-optical data to the specific dielectric increment measured in the high frequency range; both parameters appear to be molecular weight independent. The electric polarizability obtained from the Kerr effect would be originated in the induced dipoles caused by the delocalization of the bound counterions along rigid subunits whose length seems however to differ from the persistence length.     

Peptide and protein analysis by electrospray ionization-mass spectrometry and capillary electrophoresis-mass spectrometry

The extension of mass spectrometry to high molecular weight biopolymers based upon electrospray ionization and the on-line combination with capillary electrophoresis is described. Electrospray ionization produces gas-phase intact multiply charged molecular ions of biomolecules from highly charged liquid droplets by a high electric field. For high molecular weight substances electrospray ionization results in a characteristic bell-shaped distribution of multiply charged ions, with each adjacent major peak in the spectrum differing by one charge. Multiply charged molecular ions of proteins with molecular weights greater than 130,000 have been observed with a quadrupole mass spectrometer of limited mass-to-charge range (m/z 1700). Molecular weights can be readily determined for large proteins with accuracies in the range of +/- 0.01 to 0.05%; at least an order of magnitude further improvement appears feasible with improved techniques and instrumentation. The electrospray ionization method is sensitive, presently requiring samples in the 100 fmol to 10 pmol range for proteins. Initial results combining rapid separations by capillary zone electrophoresis with on-line mass spectrometric detection via the electrospray ionization source are demonstrated for myoglobin and other proteins and polypeptides. The potential for extension of these methods to molecular weights on the order of 10(6) is discussed.     

A simple iterative approach to parameter optimization.

Various bioinformatics problems require optimizing several different properties simultaneously. For example, in the protein threading problem, a scoring function combines the values for different parameters of possible sequence-to-structure alignments into a single score to allow for unambiguous optimization. In this context, an essential question is how each property should be weighted. As the native structures are known for some sequences, a partial ordering on optimal alignments to other structures, e.g., derived from structural comparisons, may be used to adjust the weights. To resolve the arising interdependence of weights and computed solutions, we propose a heuristic approach: iterating the computation of solutions (here, threading alignments) given the weights and the estimation of optimal weights of the scoring function given these solutions via systematic calibration methods. For our application (i.e., threading), this iterative approach results in structurally meaningful weights that significantly improve performance on both the training and the test data sets. In addition, the optimized parameters show significant improvements on the recognition rate for a grossly enlarged comprehensive benchmark, a modified recognition protocol as well as modified alignment types (local instead of global and profiles instead of single sequences). These results show the general validity of the optimized weights for the given threading program and the associated scoring contributions.     

Spatial Separation of Molecular Conformers and Clusters

Gas-phase molecular physics and physical chemistry experiments commonly use supersonic expansions through pulsed valves for the production of cold molecular beams. However, these beams often contain multiple conformers and clusters, even at low rotational temperatures. We present an experimental methodology that allows the spatial separation of these constituent parts of a molecular beam expansion. Using an electric deflector the beam is separated by its mass-to-dipole moment ratio, analogous to a bender or an electric sector mass spectrometer spatially dispersing charged molecules on the basis of their mass-to-charge ratio. This deflector exploits the Stark effect in an inhomogeneous electric field and allows the separation of individual species of polar neutral molecules and clusters. It furthermore allows the selection of the coldest part of a molecular beam, as low-energy rotational quantum states generally experience the largest deflection. Different structural isomers (conformers) of a species can be separated due to the different arrangement of functional groups, which leads to distinct dipole moments. These are exploited by the electrostatic deflector for the production of a conformationally pure sample from a molecular beam. Similarly, specific cluster stoichiometries can be selected, as the mass and dipole moment of a given cluster depends on the degree of solvation around the parent molecule. This allows experiments on specific cluster sizes and structures, enabling the systematic study of solvation of neutral molecules.     

TROSY and CRINEPT: NMR with large molecular and supramolecular structures in solution

TROSY and CRINEPT are new techniques for solution NMR studies of molecular and supramolecular structures. They allow the collection of high-resolution spectra of structures with molecular weights >100 kDa, significantly extending the range of macromolecular systems that can be studied by NMR in solution. TROSY has already been used to map protein-protein interfaces, to conduct structural studies on membrane proteins and to study nucleic acid conformations in multimolecular assemblies. These techniques will help us to investigate the conformational states of individual macromolecular components and will support de novo protein structure determination in large supramolecular structures.     

Strain improvement of fungal insecticides for controlling insect pests and vector-borne diseases

Insect pathogenic fungi play an important natural role in controlling insect pests. However, few have been successfully commercialized due to low virulence and sensitivity to abiotic stresses that produce inconsistent results in field applications. These limitations are inherent in most naturally occurring biological control agents but development of recombinant DNA techniques has made it possible to significantly improve the insecticidal efficacy of fungi and their tolerance to adverse conditions, including UV. These advances have been achieved by combining new knowledge derived from basic studies of the molecular biology of these pathogens, technical developments that enable very precise regulation of gene expression, and genes encoding insecticidal proteins from other organisms, particularly spiders and scorpions. Recent coverage of genomes is helping determine the identity, origin, and evolution of traits needed for diverse lifestyles and host switching. In future, such knowledge combined with the precision and malleability of molecular techniques will allow design of multiple pathogens with different strategies and host ranges to be used for different ecosystems, and that will avoid the possibility of the host developing resistance. With increasing public concern over the continued use of synthetic chemical insecticides, these new types of biological insecticides offer a range of environmental-friendly options for cost-effective control of insect pests.     

Carrier design: biodistribution of branched polypeptides with a poly(L-lysine) backbone

The biodistribution has been examined in mice of a range of synthetic branched polypeptides which are based on a polylysine backbone but which differ in ionic charge, side-chain structure, and molecular size. Polycationic polypeptides, regardless of their size or primary structure at the branches, were cleared rapidly from the circulation, the liver being the major site of clearance. Polypeptides with glutamic acid in the side chain, which would be amphoteric under physiological conditions, showed a significantly prolonged blood survival, and this was seen with polypeptides in the range of molecular weights of 46,000 up to 213,000. Such polypeptides provide a useful system with which to investigate the effect of structural parameters on the pharmacokinetic properties of carrier molecules and would allow the selection of candidate carriers for a variety of uses.     

Molecular tools in marine ecology

Molecular tools have diverse applications in marine ecology. In microbial systems, DNA sequences of rRNA and other genes have identified a variety of novel lineages of bacteria inhabiting marine environments that have resisted traditional culture methods. However, relatively few natural populations have been characterized due to the rather labor-intensive methodologies employed. Recent technological developments such as in situ PCR and flow cytometry promise to greatly enhance the speed at which microbial taxa can be identified and enumerated in field collected water and substrate samples; such advances will allow future work to employ the spatial and temporal field sampling required to monitor the impact of natural and anthropogenic changes in the environment. This approach also holds promise for examining physiological status of field collected cells, garnering information on such elusive parameters as growth rates and the extent of nutrient limitation under natural conditions. Studies of macrobiota have similarly benefited from the use of molecular approaches to species identification. This has been particularly true with regard to distinguishing among larval forms of closely related taxa which are nearly identical morphologically. Genetic variation within species assayed by molecular tools has been useful in examining the stability of populations through time and in assessing patterns of recruitment to geographically separated populations. Enhanced understanding of these ecological problems will also require intensive spatial and temporal monitoring of both larval and adult populations. Often, the newer techniques based on DNA sequence variation have practical advantages over allozyme techniques: e.g., PCR allows assay of minute quantities of DNA that may come from ethanol preserved samples. However, when ample allozyme variation exists to address a given issue, these older techniques may be favored on a variety of criteria, including speed and cost. Hence, choice of methodology should be based on the expected efficiency of a given approach to a specific problem rather than the apparent sophistication of the method itself.     

Optimized conditions for pulsed field gel electrophoretic separations of DNA.

Quantitative measurement of DNA migration in gel electrophoresis requires precisely controlled homogeneous electric fields. A new electrophoresis system has allowed us to explore several parameters governing DNA migration during homogeneous field pulsed field gel (PFG) electrophoresis. Migration was measured at different switch times, temperatures, agarose concentrations, and voltage gradients. Conditions which increase DNA velocities permit separation over a wider size range, but reduce resolution. We have also varied the angle between the alternating electric fields. Reorientation angles between 105 degrees and 165 degrees give equivalent resolution, despite significant differences in DNA velocity. Separation of DNA fragments from 50 to greater than 7000 kilobases (Kb) can easily be optimized for speed and resolution based on conditions we describe.     

An angle-variable three-dimensional pulsed field gel electrophoresis system

A three-dimensional pulsed field electrophoretic method based on the simultaneous application of fixed and cyclically alternating polarity fields at a right angle is described. Requiring only minimal electronic hardware it provides highly homogeneous field conditions over a large gel area and the versatility to vary the pulse vector angle. The electrophoretic parameters critical to achieve fast high resolution separation over a wide range of molecular sizes have been optimized and applied to megabase-size chromosomal DNA molecules. The empirical relationships between pulse time, field strength conditions, and resolution limits derived allow selection of coordinated experimental conditions for the separation of specific DNA size ranges.     

Protein blotting with direct blotting electrophoresis

Direct blotting electrophoresis, a method designed to be of general application for the separation and electroblotting of macromolecules, has been adapted to produce protein blots suitable for subsequent processing by standard techniques such as dye staining or immunological detection. After their separation in a very short gel the protein bands are electrophoresed out of the gel onto an immobilizing matrix. The matrix which is moved across the bottom of the gel by a conveyor belt binds these proteins with high affinity. Once the protein samples have been loaded onto the gel and electrophoresis has been started, no further intervention is needed until the blot is completed. The total expenditure of time for such a direct blot is less than 4 h for a mixture of proteins in the molecular weight range of 14-70 kDa. The staining sensitivity of directly blotted proteins is about 200 ng protein per band as revealed by India ink staining.