Secondary pulsed field gel electrophoresis: a new method for faster separation of larger DNA molecules.

A novel technique, which we call secondary pulsed field gel electrophoresis (SPFG) has been developed. In SPFG, short pulses are applied in the direction of net migration of the DNA in addition to the reorienting pulses used in conventional pulsed field electrophoresis (PFG). Experimental results show that SPFG extends and improves the electrophoretic resolution of DNA for molecules from 0.5 megabase pairs to over 10 megabase pairs in size. This improved resolution is obtained with dramatically shorter run times. Thus SPFG appears to circumvent a number of the key limitations in previous PFG protocols.     

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.     

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.     

Restriction mapping of recombinant lambda DNA molecules using pulsed field gel electrophoresis

We have integrated pulsed field gel electrophoresis with the partial digestion strategy of Smith and Birnstiel (1976, Nucleic Acids Res. 3,2387-2398) to generate a rapid and accurate method of restriction endonuclease mapping recombinant lambda DNA molecules. Use of pulsed field gels dramatically improves the accuracy of size determination and resolution of DNA restriction fragments relative to standard agarose gels. Briefly, DNA is partially digested with restriction enzymes to varying extents and then hybridized with a radiolabeled oligonucleotide which anneals specifically to one of the lambda cohesive (cos) ends, effectively end labeling only those digestion products containing that cos end. In this study, we have used an oligonucleotide hybridizing to the right cos end. DNA is then fractionated by pulsed field gel electrophoresis, the gel dried down, and cos end containing fragments visualized by autoradiography. Fragment sizes indicate the distances from the labeled cos end to each restriction site for the particular restriction enzyme employed. This procedure requires only minimal quantities of DNA and is applicable to all vectors utilizing lambda cos ends.     

A simple and sensitive pulsed field gel electrophoresis protocol to study 50 kb apoptotic DNA fragmentation in human lymphocytes

DNA fragmentation of 50 kb is observed in apoptotic human lymphocytes as measured with pulsed field gel electrophoresis (PFGE). Standard PFGE assay involves embedding of cells into agarose blocks followed by lysis in the presence of proteinase K. In this study, we modified the PFGE protocol by omitting the proteinase K. In this study, we modified the PFGE assay by omitting the proteinase K and changing lysis solution according to the method of anomalous viscosity time dependence (AVTD). The conditions of PFGE were adjusted aiming to compress apoptotic fragments, increasing sensitivity and the number of samples that could be loaded on the same gel. Lymphocytes were irradiated with gamma-rays and apoptotic fragmentation of DNA was determined by PFGE using standard lysis with proteinase K and lysis protocol from AVTD method. Both protocols of lysis resulted in the same pattern of DNA fragments. The yield of radiation-induced apoptotic fragmentation was higher with the AVTD protocol of lysis. The novel PFGE protocol is simple and relatively non-expensive, requires only 7 h running time and gives a possibility to analyse simultaneously up to 69 samples in the same gel. The sensitivity of our protocol provides reproducible detection of 50 kb fragmentation after irradiation of human lymphocytes with 5 cGy of gamma-rays. In 2 of 6 donors tested, this DNA fragmentation was detected at dose on 2 cGy. The novel protocol can be used for quantification of 50 kb apoptotic fragments induced by different agents including low dose ionising radiations, chemicals and electromagnetic fields.     

A model for the separation of large DNA molecules by crossed field gel electrophoresis.

The idea that large DNA molecules adopt a stretched conformation as they pass through gels suggests a simple mechanism for the separation of DNA by crossed field electrophoresis: at each change in field direction a DNA molecule takes off in the new direction of the field by a movement which is led by what was formerly its back end. The effect of this ratcheting motion is to subtract from the DNA molecule's forward movement, at each step, an amount which is proportional to its length. We find that this model explains most of the features of the separation, and we describe experiments, using a novel electrophoresis apparatus, which support the model. The apparatus turns the gel between two preset orientations in a uniform electric field at preset time intervals. This separation method has the practical advantage over some others that the DNA molecules follow straight tracks. A further advantage is that the parameters which determine the separation are readily predicted from the simple theory describing their motion.     

Separation of megabased-sized DNA molecules of Aspergillus niger using pulsed field gel electrophoresis

In this study, the chromosomal DNAs were extracted from Aspergillus niger Z10 wild type strain and these DNAs were separated using the contour clamped homogeneous electric field gel electrophoresis (CHEF) system. This system is laboratory-made and is operated by a computer program. Total DNAs resolved into five distinct chromosomal bands. The size of the chromosomes was estimated as being between 3.3 Mb to 6.4 Mb.     

The human thyroglobulin gene is over 300 kb long and contains introns of up to 64 kb.

Thyroglobulin (Tg), the precursor of thyroid hormones, is a 660.000 Da dimeric glycoprotein synthesized exclusively in the thyroid gland. We have cloned the human thyroglobulin gene from cosmid and phage libraries and constructed a complete restriction map. The gene encodes an 8.7 kb mRNA, covers at least 300 kb DNA and contains at least 37 exons separated by large introns of up to 64 kb. A striking difference in structure between the 5' and 3' part of the gene suggests that it is composed of two evolutionarily different regions. The first 30 kb DNA encode 3 kb of the mRNA, yielding an exon:intron ratio of 1:10, whereas the remaining 270 kb encodes 5.7 kb of the mRNA with an exon:intron ratio of 1:47. In thyroid cells, the Tg gene is not rearranged and nuclear RNA homologous with sequences internal to the 64 kb intron is present, suggesting that the Tg gene is transcribed as a 300 kb RNA.     

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.     

Real-time imaging of the reorientation mechanisms of YOYO-labelled DNA molecules during 90 degrees and 120 degrees pulsed field gel electrophoresis.

Pulsed field gel electrophoresis (PFGE) techniques have been developed to overcome the limitations of conventional electrophoresis and to increase the separation to DNA chromosomes of few megabase pairs in size. Despite of the large success of these techniques, the various separation protocols employed for PFGE experiments have been determined empirically. However, a deep understanding of the molecular mechanisms of motion responsible for DNA separation becomes necessary for the rational optimization of these techniques. This paper shows the first clear observations of individual molecules of DNA during the reorientation process in 90 degrees PFGE and 120 degrees PFGE. Real-time visualization of the DNA dynamics during PFGE was possible with the use of an epi-illumination fluorescence microscope specifically equipped to run these experiments and by staining the DNA with YOYO-1 (1,1'-(4,4,7,7-tetramethyl-4,7-diazaundecamethylene)-bis-4-[3-meth yl -2,3-dihydro-(benzo-1,3-oxazole)-2-methyl-idene]-quinolinium tetraiodide). This dye forms a very stable, highly fluorescent complex with double-stranded DNA and dramatically improves the quality of the DNA images. The results of computer simulations used to reproduce the molecular mechanisms of motion as well as the DNA separation features are also discussed.     

Technical improvement to prevent DNA degradation of Leptospira spp. in pulsed field gel electrophoresis

Leptospirosis is a public health problem. Infection with pathogenic Leptospira occurs by exposure to many environments and is traditionally associated with occupational risk activities. Pulsed-field gel electrophoresis was used to investigate the epidemiological relatedness among Leptospira isolates. However, analysis by PFGE yielded inconclusive data as a result of extensive DNA degradation. This degradation can be significantly reduced by the inclusion of thiourea in the electrophoresis buffer, improving the analysis of DNA banding patterns.     

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.