Field inversion gel electrophoresis with different pulse time ramps.

The influence of different pulse time ramps on the separation of yeast chromosomes with field inversion gel electrophoresis (FIGE) was investigated by the means of two dimensional gel electrophoresis. The problem of band inversion, which makes it difficult to distinguish DNA molecules of different size, has been solved by using double randomized pulse times. A major disadvantage of the field inversion technique is thereby overcome, making this system comparable to other pulsed field techniques.     

Conformational correlatives of DNA band compression and bidirectional migration during field inversion gel electrophoresis, detected by quantitative video epifluorescence microscopy.

Individual DNA molecules in the Mb size range were monitored by epifluorescence video microscopy during field inversion gel electrophoresis (FIGE). DNA migrating in an agarose gel gives rise to characteristic V-conformational elements and when doing so exhibits a reduced mobility. When the V-conformational elements per DNA molecule are few, the degree of retardation appears proportional to the number of V's, and since larger DNA species exhibit more V's, to DNA size. For a particular pulse frequency, the proportionality breaks down progressively as the number of V-conformational elements per DNA molecule increases. The loss of proportionality between DNA length and migration rate is being correlated with the macroscopically observed loss of electrophoretic size discrimination known as band compression. For a particular pulsing frequency and size class of DNA, the loss of size discrimination is thought to be due to the different orientations of migration, caused by the asymmetric distribution of V-conformational elements when the number of these elements is moderate. Small and very large DNA by contrast migrate with the direction of the biased field. These events, analyzed by microscopic measurement, are consistent with the known macroscopically observed double-valued mobilities in FIGE.     

Model and computer simulations of the motion of DNA molecules during pulse field gel electrophoresis

A model is presented for the motion of individual molecules of DNA undergoing pulse field gel electrophoresis (PFGE). The molecule is represented by a chain of charged beads connected by entropic springs, and the gel is represented by a segmented tube surrounding the beads. This model differs from earlier reptation/tube models in that the tube is allowed to leak in certain places and the chain can double over and flow out of the side of the tube in kinks. It is found that these kinks often lead to the formation of U shapes, which are a major source of retardation in PFGE. The results of computer simulations using this model are compared with real DNA experimental results for the following cases: steady field motion as seen in fluorescence microscopy, mobility in steady fields, mobility in transverse field alternation gel electrophoresis (TFAGE), mobility in field inversion gel electrophoresis (FIGE), and linear dichroism (LD) of DNA in agarose gels during PFGE. Good agreement between the simulations and the experimental results is obtained.     

Study of large DNA fragments in agarose gels by transient electric birefringence

The pulsed-field gel electrophoresis (PFG) is a newly developing technique used in the fractionation of large DNA fragments. Advances in PFG demand a better understanding in the corresponding mechanisms of DNA dynamics in the gel network. Detailed experiments are needed to verify and to extend existing theoretical predictions as well as to find optimum conditions for efficient separation of large DNA fragments. In the present study, deformation of large DNA fragments (40-70 kilobase pairs) imbedded in agarose gels were investigated by using the transient electric birefringence (TEB) technique under both singular polarity and bipolarity electric pulses at low applied electric field strengths (E less than or equal to 5 V/cm). The steady-state optical retardation (delta s) of DNA molecules is linearly proportional to E2. At a given E, the amplitude of optical retardation [delta(t)] increases monotonically with the pulse width (PW) and then reaches a plateau value [delta(t = 0) = delta s] where t = 0 denotes the time when the applied field is turned off or reversed. The field-free decay time (tau-a few minutes) is several orders of magnitudes slower than that from previous TEB observations using high electric field strengths (E-kV/cm) and short pulse widths (PW-ms). The degree of deformation (stretching and orientation) and the time of restoration to the equilibrium conformation of overall DNA chains have been related to delta and tau. In field inversion measurements, exponentially rising and linearly falling of birefringence signals in the presence of forward/inverse applied fields were observed. The rising and falling of birefringence signals were reproducible under a sequence of alternating pulses. Comparison of our results with literature findings and discussions with theories are presented.     

A systematic study of field inversion gel electrophoresis.

The mobilities of oligomers of phage lambda DNA and of yeast chromosomes in agarose gels during field inversion gel electrophoresis (FIGE) were measured at different pulse times and electric fields. Also the ratios between forward and backward pulse times and/or field gradients were varied. The problem of 'band inversion' during FIGE, leading to an ambiguity in the mobility of large DNA fragments, was solved by using two dimensional gel electrophoresis with different parameters in the first and second dimension. The results are compared with those obtained with other pulsed electrophoresis systems and with a theoretical model.     

Transient orientation of linear DNA molecules during pulsed-field gel electrophoresis.

The transient orientation of lambda DNA and lambda-DNA oligomers has been measured during pulsed field gel electrophoresis. The DNA becomes substantially aligned parallel to the electric field E. In response to a single rectangular pulse, orientation shows an overshoot with a peak at 1 second, then a small undershoot, and finally a plateau. When the field is turned off, the orientation dissipates in two distinct exponential phases. Field inversion leads to periods of orientation with intervening periods of reduced orientation as the chains reverse direction. Field inversion pulses applied to linear oligomers of lambda-DNA show that orientation responses slow down but increase in amplitude as molecular weight increases, for a given field. Because DNA stretching and alignment parallel to E are expected to correlate with DNA velocity, the velocity in response to a pulsed field is also expected to exhibit an overshoot.     

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.     

Physical characterisation of the plastid DNA in Neospora caninum

The physical characteristics of the plastid DNA in Neospora caninum were investigated using pulsed-field gel electrophoresis and TEM. In a comparison of contour-clamped homogenous electric field and field inversion gel electrophoresis, the latter proved the more successful technique for studying the plastid molecules. In most cases, restriction or modifying enzymes were required to enable the plastid DNA molecules to enter the gel from the well area. The unit length of the plastid of N. caninum is approximately 35 kb; however, there is evidence for the formation of oligomeric molecules, which may migrate as linear molecules in approximate multiples of the unit length. Four different plastid genes encoding the ssrRNA, lsrRNA, rpoC and tufA genes were identified by hybridisation studies of contour-clamped homogenous electric field and field inversion gel electrophoresis gels. Transmission EM was performed on isolated plastid DNA, and circular structures similar in size and appearance to those described in other apicomplexans were observed, with an approximate length of 19 microm. The data presented here conclusively show that the Nc-Liverpool canine strain of N. caninum possesses a plastid DNA, with physical characteristics similar to the plastids found in other apicomplexans.     

Circular and linear DNA molecules in the Entamoeba histolytica complex molecular karyotype

Entamoeba histolytica genome was analysed by pulsed field gel electrophoresis under conditions to separate linear chromosomes in the 170–1400 kb range. We identified linear DNA molecules of 227, 366, 631, 850, 1112 and 1361 kb (mean sizes obtained by three different methods) and we estimated their reorientation times and migration velocities at various experimental conditions. DNA shift mobility assays, using ethidium bromide, suggested that bands migrating at 227 and 631 kb contain linear and circular DNA, whereas a band at 436 kb has only circular DNA. We obtained a regression equation relating sizes of supercoiled DNA molecules with their migration velocities during a pulse at constant electric field and temperature. We also developed a computer program (EHPATTERNS) that predicts the migration per pulse and the resolution order of circular and linear E. histolytica DNA at different pulse times and constant driving and frictional forces. The simulation showed that linear DNA molecules frequently co-migrate with circular molecules, but circular molecules change when the pulse time varies. This molecular mixture generates broad bands and difficulties in the interpretation of the molecular karyotype of E. histolytica. Received: 19 January 1999 / Revised version: 3 November 1999 / Accepted: 22 November 1999     

Imaging of kinked configurations of DNA molecules undergoing orthogonal field alternating gel electrophoresis by fluorescence microscopy

The dynamics of individual DNA molecules undergoing orthogonal field alternating gel electrophoresis (OFAGE) have been studied by use of T2 DNA molecules labeled with a dye and visualized with a fluorescence microscope. The mechanism of reorientation used by a molecule to align itself in the direction of the new orthogonal field depends on the degree of extension of the chain immediately before the application of this field. The formation of kinks is promoted when time is allowed between the application of the two orthogonal fields so that the molecule attains a partially relaxed configuration. In this case, the chain appears bunched up in domains moving along the contour of the molecule. These regions are found to be the locations where the kinks are formed upon application of the second field perpendicular to the chain. The formation of kinks provides a significant retardation of the reorientation of the molecules, relative to molecules that do not form kinks, and appears to play an important role in the fractionation attained with OFAGE. A classification of various reorientation mechanisms observed in molecules that form kinks is presented.     

Analysis of Paramecium Macronuclear DNA Using Pulsed Field Gel Electrophoresis

.We have analyzed the macronuclear DNA of Paramecium tetraurelia using orthogonal-field-altemation gel electrophoresis. The mean size of the linear macronuclear DNA molecules is approximately 450 kb. Less than 6% of the macronuclear DNA is larger than 800 kb. Using pulse times of 20, 40, 60 and 90 s we show that the macronuclear fragment containing the A type variable surface antigen gene migrates reproducibly as a 320-kb linear DNA. Over the same pulse times we describe the unusual migration of the ribosomal RNA gene (rDNA) of P. tetraurelia. At pulse times of 20 and 40 s the rDNA migrates at limit mobility (300 and 500 kb, respectively) whereas with 60- and 90-s pulse times, 2 components of rDNA are observed; 1 fraction independent of pulse time migrating at limit mobility, and a 2nd component migrating between 100-kb and 400-kb linear markers. Based upon previous electron micrographic studies of Paramecium rDNA as well as data presented here we conclude that the majority of Paramecium rDNA molecules are a circular DNA form.     

High-resolution separation and accurate size determination in pulsed-field gel electrophoresis of DNA. 4. Influence of DNA topology

Pulsed-field gel electrophoresis is a powerful technique for the fractionation of linear DNA molecules with sizes above 50 kilobase pairs (kb). Here it is demonstrated that this technique is also effective for separating smaller DNAs including linear, circular, and supercoiled species. The mobilities of linear DNAs larger than 8 kb can be modulated by pulse times between 0.1 and 100 s. The mobility of supercoiled DNA molecules up to 16 kb is generally unaffected by these pulse times except that 10-s pulse times cause a small but distinct increase in the mobility. The general insensitivity of small supercoiled DNAs to pulse time presumably occurs because these species reorient so rapidly that they spend most of their time undergoing conventional electrophoresis. However, the mobilities of larger supercoiled DNAs are affected by pulse times of less than 1 s, and at 0.1 s the molecules are better resolved by pulsed electrophoresis than by ordinary electrophoresis. The mobility of 3-19 kb nicked and relaxed circular DNA molecules is also affected by pulse time but in a complex way.     

Separation of chromosomal DNA molecules from C.albicans by pulsed field gel electrophoresis.

Modifications have been made to standard pulse field gel electrophoresis (PFGE) systems to enable very large DNA molecules to be resolved. The single most important modification was to elevate the temperature of electrophoresis to 35 degrees C. This enabled the largest Saccharomyces cerevisiae chromosome to be reproducibly resolved. More impressively, it enabled the DNA of Candida albicans to be clearly resolved into six bands, a feat which was very difficult at lower temperatures. Even so, optimal resolution could only be obtained by carefully adjusting field voltages and switching times. The DNA from the two largest C. albicans chromosomes, which was estimated to be at least 5-10Mbp in size, ran somewhat anomalously, giving fuzzy bands which did not migrate in the direction of the average electric field. That the highest molecular weight band was a distinct chromosome was demonstrated by specific hybridisation to the C. albicans ADE2 gene probe. With further fine tuning, the PFGE system described here should be capable of resolving DNA from the smallest human chromosomes.     

Pulse time and agarose concentration affect the electrophoretic mobility of cccDNA during PFGE and FIGE [corrected].

Circular DNAs have been shown to migrate in an unusual manner during field inversion gel electrophoresis (FIGE) and orthogonal field alternating gel electrophoresis (OFAGE). We studied the effect of varying pulse time and agarose concentration on the electrophoretic mobility of supercoiled (ccc) DNAs ranging from 2 kbp to 16 kbp during FIGE and contoured homogeneous electric fields (CHEF). Both supercoiled and linear molecules display a minimum mobility as a function of pulse time in a CHEF apparatus. Linear and cccDNAs of the same size are differently affected by pulse time. Pulse-time dependence was observed for cccDNAs in both systems. Pulse-time dependence in FIGE is very small at a 1.0% agarose concentration, but is pronounced in 0.8% or 1.2% gels.     

Electrophoresis of DNA in oriented agarose gels

Oriented agarose gels were prepared by applying an electric field to molten agarose while it was solidifying. Immediately afterwards, DNA samples were applied to the gel and electrophoresed in a constant unidirectional electric field. Regardless of whether the orienting field was applied parallel or perpendicular to the eventual direction of electrophoresis, the mobilities of linear and supercoiled DNA molecules were either faster (80% of the time) or slower (20% of the time) than observed in control, unoriented gels run simultaneously. The difference in mobility in the oriented gel (whether faster or slower) usually increased with increasing DNA molecular weight and increasing voltage applied to orient the agarose matrix. In perpendicularly oriented gels linear DNA fragments traveled in lanes skewed toward the side of the gel; supercoiled DNA molecules traveled in straight lanes. If the orienting voltage was applied parallel to the direction of electrophoresis, both linear and supercoiled DNA molecules migrated in straight lanes. These effects were observed in gels cast from different types of agarose, using various agarose concentrations and two different running buffers, and were observed both with and without ethidium bromide incorporated in the gel. Similar results were observed if the agarose was allowed to solidify first, and the orienting electric field was then applied to the gel for several hours before the DNA samples were added and electrophoresed. The results suggest that the agarose matrix can be oriented by electric fields applied to the gel before and probably during electrophoresis, and that orientation of the matrix affects the mobility and direction of migration of DNA molecules. The skewed lanes observed in the perpendicularly oriented gels suggest that pores or channels can be created in the matrix by application of an electric field. The oriented matrix becomes randomized with time, because DNA fragments in oriented and unoriented gels migrated in straight lanes with identical velocities 24 hours later.     

Separation of large DNA molecules by modified pulsed field gradient gel electrophoresis

Resolution of DNA fragments by pulsed field gradient gel electrophoresis is a function of the pulse time, geometry, and strength of the orthogonal electric fields. The first field geometry described had a number of disadvantages. We show that these disadvantages can be largely overcome by a modified electric field geometry together with an altered switch pattern. These changes are shown to have critical consequences for the technique. Resolution is more uniform across the gel, which permits more samples to be analyzed on the same gel. In addition, DNA molecules follow a migration path that is approximately straight down the gel. This aspect also increases the number of usable wells. One important property of the system described here provides some insight into the mechanism whereby DNA molecules are resolved by this method.     

BBC microcomputer controlled field inversion gel electrophoresis

Agarose gel electrophoresis to separate DNA molecules is a widelyused technique in molecular biology but there is an upper limitto the sizes that can be resolved. Pulsed field techniques haveextended this limit but require expensive equipment. Here wedescribe a home-made control unit to interface conventionalelectrophoresis equipment to a BBC microcomputer for the purposesof field inversion gel electrophoresis. Received on October 6, 1987; accepted on November 10, 1987     

The mobility minima in pulsed-field capillary electrophoresis of large DNA.

Pulsed-field capillary electrophoresis represents a new tool for rapid and highly efficient separations of large biopolymers. The method has been utilized here to study dependencies of the electrophoretic mobility upon the frequency and pulse shape of applied voltage for large, double-stranded DNA molecules (5-100 kb) migrating in neutral polymer solutions. Two different shapes of alternating electric field (sine- and square-wave impulses) were examined with the frequency values ranging from 1 to 30 Hz. The linear dependence between duration of the forward pulse (at which the DNA molecule experiences a minimum mobility) and the product N.In(N) (where N is the number of base pairs) was experienced in field-inversion gel electrophoresis, while exponential dependence was found with the sinusoidal electric field. The mobility minima were lower in field-inversion electrophoresis than with the biased sinusoidal-field technique. The DNA (5 kb concatamers) was adequately separated using a ramp of frequency in the square-wave electric field, in approximately 1 h. The migration order of DNA fragments was referenced through adding a monodisperse DNA (48.5 kb) into the sample. The band inversion phenomena were not observed under any experimental conditions used in this work.     

Plasmid migration using orthogonal-field-alternation gel electrophoresis.

The migration properties of a series of supercoiled plasmids ranging in size from 4 to 16 kilobases (kb) have been analyzed by orthogonal-field-alternation gel electrophoresis (OFAGE). These circular DNAs enter the gel and are well resolved. Unlike linear DNA molecules, the relative mobilities of these plasmids are constant over a wide range of pulse times, from 10 to 120 seconds, as well as over a broad range of total running times, from 6 to 24 hours. Electrophoresis of supercoiled, relaxed, and nicked open circular forms as well as topoisomers of pBR322 shows that the extent of supercoiling has a dramatic effect on plasmid migration on OFAGE. Several practical applications for exploiting the different migration properties of circular and linear DNA molecules on OFAGE are presented.     

Measurement of radiation-induced DNA double-strand breaks by pulsed-field gel electrophoresis

We have examined the use of pulsed-field gel electrophoresis (PFGE) to measure DNA double-strand breaks induced in CHO cells by ionizing radiation. The PFGE assay provides a simple method for the measurement of DNA double-strand breaks for doses as low as 3-4 Gy ionizing radiation, and appears applicable for the measurement of damage produced by any agent producing double-strand breaks. The conditions of transverse alternating field electrophoresis determined both the sensitivity of the assay and the ability to resolve DNA fragments with different sizes. For example, with 0.8% agarose and a 1-min pulse time at 250 V for 18 h of electrophoresis, 0.39% of the DNA per gray migrated into the gel, and only molecules less than 1500 kb could be resolved. With 0.56% agarose and a 60-min pulse time at 40 V for 6 days of electrophoresis, 0.55-0.90% of the DNA per gray migrated into the gel, and molecules between 1500 and 7000 kb could be resolved.