Contour-clamped homogeneous electric field electrophoresis of Staphylococcus aureus

Contour-clamped homogeneous electric field (CHEF) electrophoresis is a technique of pulsed-field gel electrophoresis that enables the resolution of large fragments of DNA that cannot be resolved by conventional gel electrophoresis. The procedure involves the application of controlled electric fields that change direction at a predetermined angle to samples of DNA that have been embedded in an agarose gel matrix and digested with a restriction endonuclease. Adjustment of the electrophoresis conditions enables the separation of DNA fragments with lengths from 10 kilobases up to 9 megabases in a size-dependent manner in agarose gels. The banding patterns can be used for epidemiological typing, the separated DNA can be immobilized onto a membrane and used for genetic mapping, or individual fragments can be extracted and used for downstream genetic manipulations. The protocol requires specialized equipment and can be completed in a maximum of 7 days.     

A DNA prism for high-speed continuous fractionation of large DNA molecules

The analysis and fractionation of large DNA molecules plays a key role in many genome projects. The standard method, pulsed-field gel electrophoresis (PFGE), is slow, with running times ranging from 10 hours to more than 200 hours. In this report, we describe a thumbnail-sized device that sorts large DNA fragments (61-209 kilobases (kb)) in 15 seconds, with a resolution of approximately 13%. An array of micron-scale posts serves as the sieving matrix, and integrated microfluidic channels spatially shape the electric fields over the matrix. Asymmetric pulsed fields are applied for continuous-flow operation, which sorts DNA molecules in different directions according to their molecular masses, much as a prism deflects light of different wavelengths at different angles. We demonstrate the robustness of the device by using it to separate large DNA inserts prepared from bacterial artificial chromosomes, a widely used DNA source for most genomics projects.     

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.     

High-resolution separation and accurate size determination in pulsed-field gel electrophoresis of DNA. 2. Effect of pulse time and electric field strength and implications for models of the separation process

Bacteriophage DNAs annealed into linear oligomeric concatemers were used to examine the quantitative pulsed-field gel electrophoretic behavior of different-sized DNAs as a function of electrical field strength and pulse time. Three zones of resolution are observed for increasingly larger DNAs. In the first two zones, the electrophoretic mobility decreases linearly with increasing DNA size. The separation in zone 2 is roughly twice that in zone 1. The largest DNA molecules do not resolve at all and migrate in a compression zone. Mobility in zone 1 increases linearly with the electric field strength and decreases with the inverse of the pulse time. The behavior of DNA in zone 2 is qualitatively similar. However, the effect of field strength and pulse time on the separations in each zone is quite different. The results for zone 1 are generally consistent with the predictions of several existing physical models of pulsed-field gel electrophoresis, but no model accounts for all of the observed behavior in the three zones.     

Pulsed-field gel electrophoresis supports the presence of host-adapted Salmonella enterica subsp. enterica serovar Typhimurium strains in the British garden bird population

Salmonellosis is a frequently diagnosed infectious disease of passerine birds in garden habitats within Great Britain with potential implications for human and domestic animal health. Postmortem examinations were performed on 1,477 garden bird carcasses of circa 50 species from England and Wales, 1999 to 2007 inclusive. Salmonellosis was confirmed in 263 adult birds of 10 passerine species in this 11-year longitudinal study. A subset of 124 fully biotyped Salmonella enterica subsp. enterica serovar Typhimurium isolates was examined using pulsed-field gel electrophoresis to investigate the hypothesis that these strains are host adapted and to determine whether this molecular technique offers greater resolution in understanding the epidemiology of Salmonella Typhimurium infection than phage typing alone. For the two most common phage types, definitive type (DT) 40 and DT56v, which together accounted for 97% (120/124) of isolates, pulsed-field gel electrophoresis groupings closely correlated with phage type with remarkably few exceptions. A high degree of genetic similarity (>90%) was observed within and between the two most common pulsed-field gel electrophoresis groups. No clustering or variation was found in the pulsed-field gel electrophoresis groupings by bird species, year, or geographical region beyond that revealed by phage typing. These findings support the hypothesis that there are currently two host-adapted Salmonella phage types, S. Typhimurium DT40 and DT56v, circulating widely in British garden birds and that the reservoir of infection is maintained within wild bird populations. Large-scale multilocus sequence typing studies are required to further investigate the epidemiology of this infection.     

Pulsed-field electrophoresis of megabase-sized DNA.

Success in constructing a physical map of the human genome will depend on two capabilities: rapid resolution of very large DNA and identification of migration anomalies. To address these issues, a systematic exploration of pulsed-field electrophoresis conditions for separating multimegabase-sized DNA was undertaken. Conditions were found for first liberating and then separating DNA up to 6 megabases at higher field strengths and more rapidly than previously reported. In addition, some conditions for transversely pulsed fields produced mobility inversion, in which increased size was accompanied by faster rather than slower migration. Importantly, anomalous migration could be identified by the presence of lateral band spreading, in which the DNA band remained sharply defined but spread laterally while moving down the gel. These results have implications for both practical applications and theoretical models of pulsed-field electrophoresis.     

High-resolution separation and accurate size determination in pulsed-field gel electrophoresis of DNA. 1. DNA size standards and the effect of agarose and temperature

Pulsed-field gel electrophoresis (PGF) subjects DNA alternately to two electrical fields to resolve DNA ranging from 10,000 base pairs (10 kb) to 10,000 kb in size. The separations are quite sensitive to a variety of experimental variables. This makes it critical to have a wide range of reliable size standards. A technique is described for preparing mixtures of bacteriophage DNA oligomers that span a size range from monomer to more than 30-mer. The relationship between size and mobility of oligomers of different bacteriophage DNA monomers is generally self-consistent. Thus, these samples can serve as primary length standards for DNAs ranging from 10 kb to more than 1500 kb. They have been used to estimate the size of the chromosomal DNAs from various Saccharomyces cerevisiae strains and to test the effect of gel concentration and temperature on PFG. DNA resolution during PFG is slightly improved in agarose gels with small pore sizes, in contrast to continuous electrophoresis where the opposite is observed. PFG mobility is surprisingly sensitive to changes in the running temperature.     

Use of pulsed-field gel electrophoresis for epidemiological study of Escherichia coli O157:H7 during a food-borne outbreak.

Food and patient isolates from an Escherichia coli O157:H7 outbreak associated with undercooked ground beef were characterized by pulsed-field gel electrophoresis and Shiga-like toxin genotype. Pulsed-field gel electrophoresis confirmed the epidemiologically implicated source of the two-state outbreak and differentiated between outbreak and sporadic strains.     

The basis of high resolution separation of small DNAs by asymmetric-voltage field inversion electrophoresis and its application to DNA sequencing gels.

We have previously shown that asymmetric-voltage field inversion electrophoresis produces more uniform separation for fragments between 1 and 50 kilobases (kb) than other modes of pulsed field gel electrophoresis. We now report on the basis of this phenomenon. As in conventional electrophoresis, the pulsed field mobility of DNAs between 1 and 50 kb varies with voltage in a size dependent manner. The complex migration pattern obtained with asymmetric-voltage field inversion electrophoresis reflects the difference between the mobilities of each sized fragment under the conditions used for the forward and reverse fields. We have applied this technique to DNA sequencing gels and find improvement in resolution for single-stranded fragments in polyacrylamide gels.     

Differentiation of the subspecies of Campylobacter fetus by genomic sizing.

Campylobacter fetus subsp. fetus and C. fetus subsp. venerealis are currently differentiated by tolerance to glycine and by their epidemiology. Analysis of C. fetus DNA by pulsed-field gel electrophoresis, after digestion with the restriction endonucleases SmaI and SalI, was used to differentiate between the subspecies. All strains presently identified as C. fetus subsp. fetus had a genomic size of 1.1 Mb, whereas the majority of the C. fetus subsp. venerealis strains had a genomic size of 1.3 Mb. An additional group of strains, which were previously described as C. fetus subsp. venerealis biovar "intermedius" and were able to tolerate higher concentrations of glycine than the rest of the C. fetus subsp. venerealis strains, had an average genome size of 1.5 Mb. We suggest that pulsed-field gel electrophoresis may be useful as an additional aid in the differentiation of C. fetus strains at the subspecies level.     

Genotypic characterization of Mycobacterium avium strains recovered from animals and their comparison to human strains.

Mycobacterium avium was recovered from 21 birds and 10 pigs. Bird isolates carried IS901 and a few copies of IS1245 and appeared highly related by pulsed-field gel electrophoresis. Pig isolates showed features previously described in human isolates: a lack of IS901, a high copy number of IS1245, and marked polymorphism by pulsed-field gel electrophoresis.     

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.     

Transformation of Chlorobium limicola by a plasmid that confers the ability to utilize thiosulfate.

A modified transformation method for Chlorobium limicola confirms the role of an endogenous plasmid in thiosulfate metabolism. The plasmid was present in two forma specialis thiosulfatophilum (Tio+) strains and absent in one non-thiosulfate-utilizing (Tio-) strain. The plasmid (size, 14 kb) was transferred by transformation from Tio- to Tio+. The chromosomal restriction patterns, analyzed by pulsed-field gel electrophoresis, were used to distinguish between the different strains.     

Genetic relatedness amongst intestinal spirochaetes isolated from rate and brids.

Multilocus enzyme electrophoresis was used to determine genetic relationships amongst 32 intestinal sprrochaetes ( Serpulina spp.) isolated from rats (17), rheas (7), chickens (4), ducks (2), a swan (1) and a flamingo (1). The strains were divided into 20 electrophoretic types (ETs), with a mean genetic diversity per locus of 0.62. The results were compared with those previously published for procine intestinal spirochaetes. One strain from a healthy rat, and three rhea strains which were recovered from cases of necrotizing typhlitis, were grouped in the same ETs as certain procine strains of Serpulina hydysenteriae . The rhea strains could be differentiated from these by pulsed-field gel electrophoresis. Fifteen of the rat strains could be differentiated from these by pulsed-field gel electrophoresis. Fifteen of the rat strains were genetically and phenotypically closely related. In contrast the avian strains were genetically more heterogeneous, with pathogenic isolates located in three different genetic groups.     

Molecular cytogenetics of alpha satellite DNA from chromosome 12: fluorescence in situ hybridization and description of DNA and array length polymorphisms.

A 340-bp EcoRI fragment of alpha satellite DNA from human chromosome 12 has been isolated and used in molecular cytogenetic and genetic studies. The clone, pSP12-1, detects tandemly repeated 1.4-kb repeat units at the centromeric region of chromosome 12. By fluorescence in situ hybridization, biotinylated pSP12-1 is highly specific for chromosome 12 and has been used to confirm an i(12p) in a case of Pallister-Killian syndrome, both in metaphase spreads and in interphase nuclei. A dominant DNA polymorphism for the centromeric D12Z3 locus is detected with the enzyme TaqI. In addition, a high frequency of D12Z3 array length polymorphisms can be detected using pulsed-field gel electrophoresis. The D12Z3 array has been measured by pulsed-field gel electrophoresis to span approximately 2,250-4,300 kb at the centromeric region of chromosome 12.     

A physical map of a 1.3-Mb region on the long arm of chromosome 12, spanning the GLI and LRP loci.

We have used pulsed-field gel electrophoresis to construct a long-range restriction map spanning more than 1.3 million bp of the q13-q14 segment of chromosome 12. Within this region lie the genes coding for the gli oncogene and the low-density lipoprotein receptor-related protein (LRP). The distance between the genes is about 200-300 kb. We also observe a methylation-free island 3' to the LRP gene.     

Factors influencing DNA migration from individual cells subjected to gel electrophoresis.

Alkaline and neutral gel electrophoresis of individual mammalian cells allows detection of DNA single- and double-strand breaks, respectively. For both the alkaline and the neutral assays, lysis conditions influence how much DNA migrates, and factors in addition to DNA size play a role in migration. In particular, the tight packing of DNA in individual nuclei appears to reduce the ability to detect double-strand breaks in all of the genome. Tangling of DNA molecules is probably also responsible for the presence of "wings" associated with each nucleus after application of pulsed-field gel electrophoresis; these wings were aligned in the directions of the pulsed field, not along the resultant vector of the fields as was expected. The choice of fluorescent staining methods (propidium iodide, Hoechst 33342, or antibodies against bromodeoxyuridine) did not influence sensitivity for detecting DNA damage.     

Flash and smash: rapid freezing of muscle fibers activated by photolysis of caged ATP.

A new approach was used to study transient structural states of cross-bridges during activation of muscle fibers. Rabbit skinned muscle fibers were rapidly and synchronously activated from the rigor state by photolysis of caged ATP in the presence of Ca2+. At several different times during the switch from rigor to fully active tension development, the fibers were rapidly frozen on a liquid helium-cooled metal block, freeze-substituted, and examined in an electron microscope. The limits of structural preservation and resolution with this technique were analyzed. We demonstrate that the resolution of our images is sufficient to draw the following conclusions about cross-bridge structure. Rigor cross-bridges point away from the Z-line and most of them are wider near the thin filaments than near the backbone of the thick filaments. In contrast, cross-bridges in actively contracting fibers stretch between the thick and thin filaments at a variable angle, and are uniformly thin. Diffraction patterns computed from contracting muscle show layer lines both at 38 and 43 nm indicating that active cross-bridges contribute mass to both the actin- and myosin-based helical periodicities. The images obtained from fibers frozen 20 ms after release of ATP show a mixture of rigor and active type cross-bridge configurations. There is little evidence of cross-bridges with the rigor shape by 50 ms, and the difference in configurations between 50 and 300 ms after photolysis is surprisingly subtle.