Electrophoretic analysis of Histoplasma capsulatum chromosomal DNA.

Seven chromosome-sized DNA molecules in the Downs strain of Histoplasma capsulatum were resolved by using chromosome-specific DNA probes in blot hybridizations of contour-clamped homogeneous electric field (CHEF) and field-inversion gel electrophoresis (FIGE) agarose gels. The sizes of the chromosomal DNA bands extended from that of the largest Saccharomyces cerevisiae chromosome to beyond that of the Schizosaccharomyces pombe chromosomes. Under our experimental conditions, the order of the five largest DNA bands was inverted in the FIGE gel relative to the CHEF gel, demonstrating a characteristic of FIGE whereby large DNA molecules may have greater rather than lesser mobility with increasing size. Comparison of the Downs strain with other H. capsulatum strains by CHEF and FIGE analysis revealed considerable variability in band mobility. The resolution of seven chromosome-sized DNA molecules in the Downs strain provides a minimum estimate of the chromosome number.     

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.     

Characterization of the ''unusual'' mobility of large circular DNAs in pulsed field-gradient electrophoresis.

Large circular amplified DNAs (30 and 85 kb) present in methotrexate-resistant Leishmania major appear to migrate anomalously in pulsed field-gradient electrophoresis (PFGE), exhibiting pulse time-dependent mobility and migrating along a different apparent path relative to the large linear chromosomal DNAs. Quantitative studies indicate that the relative pulse-time dependence is actually conferred by the mobility properties of the large linear DNAs. One contributing factor to the difference in migration path is variability in the intrinsic voltage-dependence of mobility of supercoiled and linear DNAs, in combination with the asymmetrical/inhomogeneous voltage gradients. Certain linear chromosomes exhibit a previously undescribed pulse-time dependence in the voltage-dependence of mobility. When enzymatically relaxed or physically nicked the large circular DNAs fail to leave the well using any pulse time, a property also observed in conventional electrophoresis. These findings are relevant to PFGE theory, and its application to the study of circular DNA amplification in Leishmania and other species.     

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     

Pulsed field gel electrophoresis: Theory,instruments and application

Pulsed field gel electrophoresis (PFGE) is a technique for the fractionation of high-molecular-weight DNA ranging from 10 kb to 10 Mb by electrophoresis in agarose gel with an electric field that alternates (pulsates) in two directions. This technology plays a key role in modern genomics, as it allows manipulations with DNA of whole chromosomes or their large fragments. In this review, we discuss (1) the theory behind PFGE; (2) different instruments based on the principle of pulsed field, as well as their advantages and limitations; (3) factors affecting the DNA mobility in PFGE gel; and (4) practical applications of the technique.     

Pulsed-field electrophoresis indicates larger-than-expected sizes for mycoplasma genomes.

The sizes of large DNA fragments produced from genomes of members of the Mycoplasmataceae by digestion with restriction endonucleases having infrequent (1 to 3) cleavage sites within the genome were estimated from their mobility in contour-clamped homogeneous electric field (CHEF) agarose gel electrophoresis by comparison with yeast chromosomal DNA markers. The estimates of total genome size for 7 strains of 6 species ranged from approximately 900 kilo base pairs (kb) for Ureaplasma urealyticum 960T to 1330 kb for M. mycoides subsp. mycoides, GC-1176. The values derived from this new method are considerably higher than those of approximately 500 Mdaltons or 750 kb previously reported for genome sizes in members of the Mycoplasmataceae.     

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.     

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.     

Brownian Dynamics Simulation of DNA Gel Electrophoresis

Abstract

Brownian dynamics computer simulation technique was applied to investigate DNA dynamics in gel electrophoresis. Under a constant electric field of moderate strength, large DNA chains take stretched and contracted conformations alternatively during the migration. The conformation change is quasi-periodic under certain conditions, and its frequency is closely related to the experimentally-found suitable frequency of pulse field gel electrophoresis.     

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.     

Observation of DNA molecules undergoing capillary electrophoresis.

This paper presents a method to observe the motions and configurations of large DNA molecules undergoing capillary electrophoresis (CE). A simple device to perform CE horizontally under microscopic observation is designed and images of single DNA molecules inside the capillary are obtained using an epi-fluorescence microscope. DNA molecules moved towards the negative electrode when an electric field was applied. The mobilities of three types of DNA (T4 and lambda bacteriophage DNA and PBR322 plasmid DNA) were measured at different electric field strength. The mobility vs. electric field strength curves of these three large DNAs showed that the mobility remained constant at high electric field strength (200-600 Volt/cm) and increased significantly at low electric field strength (less than or equal to 50 Volt/cm.). The apparent mobilities of the large DNA molecules were independent of molecular weight. At electric field strengths greater than or equal to 400 Volt/cm., big aggregates (snowballs) of DNA molecules formed and moved upstream towards the positive electrode. When the field was turned off, the aggregates dissociated into a cloud of single DNA molecules, and diffused into the solution.     

High-resolution mapping of the gamma-aminobutyric acid receptor subunit beta 3 and alpha 5 gene cluster on chromosome 15q11-q13, and localization of breakpoints in two Angelman syndrome patients.

The gamma-aminobutyric acid (GABAA) receptors are a family of ligand-gated chloride channels constituting the major inhibitory neurotransmitter receptors in the nervous system. In order to determine the genomic organization of the GABAA receptor beta 3 subunit gene (GABRB3) and alpha 5 subunit gene (GABRA5) in chromosome 15q11-q13, we have constructed a high-resolution physical map using the combined techniques of field-inversion gel electrophoresis and phage genomic library screening. This map, which covers nearly 1.0 Mb, shows that GABRB3 and GABRA5 are separated by less than 100 kb and are arranged in a head-to-head configuration. GABRB3 encompasses approximately 250 kb, while GABRA5 is contained within 70 kb. This difference in size is due in large part to an intron of 150 kb within GABRB3. We have also identified seven putative CpG islands within a 600-kb interval. Chromosomal rearrangement breakpoints--in one Angelman syndrome (AS) patient with an unbalanced translocation and in another patient with a submicroscopic deletion--are located within the large GABRB3 intron. These findings will facilitate chromosomal walking strategies for cloning the regions disrupted by the DNA rearrangements in these AS patients and will be valuable for mapping new genes to the AS chromosomal region.     

Analysis of amplified DNAs from drug-resistant Leishmania by orthogonal-field-alternation gel electrophoresis. The effect of size and topology on mobility

Amplified extrachromosomal DNAs from antifolate-resistant Leishmania are 30-75 kilobase (kb) supercoiled molecules that resolve on orthogonal-field-alternation gel electrophoresis (OFAGE) gels. These DNAs comigrate with smaller supercoiled plasmids (7-8 kb), and their mobility is not a simple function of their size. The properties of the amplified DNAs were investigated to determine if an unusual structure accounts for the observed mobility of the amplified DNAs by OFAGE; however, their topological properties were similar to those of standard Escherichia coli plasmids. The migration of a series of supercoiled plasmids ranging in size from 6 to 91 kb was analyzed by OFAGE, and a triphasic pattern was observed. The mobilities of plasmids between 20 and 60 kb increase with size, whereas the migration of plasmids between 6 and 20 and 60 and 91 kb is inversely proportional to size. Like smaller plasmids, the large supercoiled DNAs show a pulse time-independent mobility by OFAGE. The mobility of amplified DNA from Leishmania is in accord with that of the plasmid markers. Therefore, it is primarily the size of the amplified extrachromosomal DNAs from Leishmania, rather than an unusual superhelical density or topological structure, that results in the previously unexplained migration pattern.     

Physical mapping of the Mycoplasma gallisepticum S6 genome with localization of selected genes.

We report the construction of a physical map of the Mycoplasma gallisepticum S6 genome by field-inversion gel electrophoresis of DNA fragments generated by digestion of genomic DNA with rare-cutting restriction endonucleases. The size of the M. gallisepticum S6 genome was calculated to be approximately 1,054 kb. The loci of several genes have been assigned to the map by Southern hybridization utilizing specific gene probes.     

Accurate evaluation of the sizes of DNA fragments (from 30 to 4700 kb) in pulse field gel electrophoresis.

Construction of long-range genomic maps by pulse field gel electrophoresis requires optimum resolution of large DNA fragments. Using the transverse-alternating field electrophoresis system, we describe a method to accurately evaluate the sizes of fragments generated by rare-cutter digestions within the 30-4700-kb range. A protocol generating large (greater than 1000 kb) molecules by partial digestion is also reported.     

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.     

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.     

Unidirectional pulsed-field electrophoresis of single- and double-stranded DNA in agarose gels: analytical expressions relating mobility and molecular length and their application in the measurement of strand breaks

Unidirectional pulsed-field electrophoresis improves the separation of single-stranded DNA molecules longer than 20 kilobases (kb) in alkaline agarose gels compared to static-field electrophoresis. The greatest improvement in separation is for molecules longer than 100 kb. The improved resolution of long molecules with unidirectional pulsed-field electrophoresis makes possible the measurement of lower frequencies of single-strand breaks. The analytical function that relates the length and mobility of single-stranded DNA electrophoresed with a static field also applies to unidirectional pulsed field separations. Thus, the computer programs used to measure single-strand breaks are applicable to both undirectional pulsed- and static-field separations. Unidirectional pulsed-field electrophoresis also improves the separation of double-stranded DNA in neutral agarose gels. The function relating molecular length and mobility for double-stranded DNA separated by unidirectional pulsed-field electrophoresis is a superset of the function for single-stranded DNA. The coefficients of this function can be determined by iterative procedures.     

Dependence of the electrophoretic mobility of DNA in gels on field intermittency

The electrophoretic mobility of double helical DNA in agarose and polyacrylamide gels increases as a function of time after the electric field is applied to the gel and decreases after the field is terminated. The changes are large for long (more than 10 kb) molecules. The effects of other variables are indicated.     

Quantitative analysis of the three regimes of DNA electrophoresis in agarose gels

An extensive series of experiments has been performed to study the mobility of DNA fragments ranging in size from 2.0 to 48.5 kilobose pairs. By varying the agarose concentration in the gels and the electric field strength, three DNA electrophoresis regimes were clearly identified: the Ogston regime (small DNA fragments in large pores of agarose), the reptation regime without DNA chain stretching (small pores of agarose and weak electric fields), and the reptation regime with DNA chain stretching (small pores of agarose, strong electric fields, and large DNA fragments). Here we report on the experimental identification of these regimes and on the conditions governing the transition between each of them. The onset of reptation and of stretching of DNA chains in gel electrophoresis are described quantitatively for the first time, and a phase diagram for the dynamics of DNA during electrophoresis is presented.