Trapping and breaking of in vivo nicked DNA during pulsed field gel electrophoresis

Pulsed field gel electrophoresis (PFGE) offers a high-resolution approach to quantify chromosomal fragmentation in bacteria, measured as percentage of chromosomal DNA entering the gel. The degree of separation in pulsed field gel (PFG) depends on the size of DNA as well as various conditions of electrophoresis such as electric field strength, time of electrophoresis, switch time, and buffer composition. Here we describe a new parameter, the structural integrity of the sample DNA itself, that influences its migration through PFGs. We show that subchromosomal fragments containing both spontaneous and DNA damage-induced nicks are prone to breakage during PFGE. Such breakage at single-strand interruptions results in artifactual decrease in molecular weight of linear DNA making accurate determination of the number of double-strand breaks difficult. Although breakage of nicked subchromosomal fragments is field strength independent, some high-molecular-weight subchromosomal fragments are also trapped within wells under the standard PFGE conditions. This trapping can be minimized by lowering the field strength and increasing the time of electrophoresis. We discuss how breakage of nicked DNA may be mechanistically linked to trapping. Our results suggest how to optimize conditions for PFGE when quantifying chromosomal fragmentation induced by DNA damage.     

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.     

Structure of amplified DNA, analyzed by pulsed field gradient gel electrophoresis

Pulsed field gradient electrophoresis allows the separation of large DNA molecules up to 2,000 kilobases (kb) in length and has the potential to close the resolution gap between standard electrophoresis of DNA molecules (smaller than 50 kb) and standard cytogenetics (larger than 2,000 kb). We have analysed the amplified DNA in four cell lines containing double minute chromosomes (DMs) and two lines containing homogeneously staining regions. The cells were immobilized in agarose blocks, lysed, deproteinized, and the liberated DNA was digested in situ with various restriction endonucleases. Following electrophoretic separation by pulsed field gel electrophoresis, the DNA in the gel was analysed by Southern blotting with appropriate probes for the amplified DNA. We find that the DNA in intact DMs is larger than 1,500 kb. Our results are also compatible with the notion that the DNA in DMs is circular, but this remains to be proven. The amplified segment of wild-type DNA covers more than 550 kb in all lines and possibly up to 2,500 kb in some. We confirm that the repeat unit is heterogeneous in some of the amplicons. In two cell lines, however, with low degrees of gene amplification, we find no evidence for heterogeneity of the repeats up to 750 (Y1-DM) and 800 kb (3T6-R50), respectively. We propose that amplicons start out long and homogeneous and that the heterogeneity in the repeat arises through truncation during further amplification events in which cells with shorter repeats have a selective advantage. Even if the repeats are heterogeneous, however, pulsed field gradient gels can be useful to establish linkage of genes over relatively short chromosomal distances (up to 1,000 kb). We discuss some of the promises and pitfalls of pulsed field gel electrophoresis in the analysis of amplified DNA.     

The effect of DNA concentration on mobility in pulsed field gel electrophoresis.

The effect of DNA concentration on pulsed field gel electrophoretic mobility was studied for human genomic DNA prepared in agarose inserts at 8-800 micrograms/ml and digested to completion with Not I. An eighth of each 100 microliter insert was used to produce DNA loads of 0.1 to 10 micrograms per lane. The mobility of single copy restriction fragments, as detected by hybridization, was largely concentration independent when DNA concentrations were 80 micrograms/ml or less. However, at DNA concentrations of 200 micrograms/ml and greater, dramatic effects of DNA concentration are evident. In the worst case, at 800 micrograms/ml, the apparent size of a DNA fragment is almost 2.5 times its true size. At constant DNA concentrations, increasing the DNA mass loads by loading larger insert slices had no further effect on DNA electrophoretic mobility, although the bands were broader for bigger insert slices. Thus, for precise and accurate sizing in pulsed field gel electrophoresis the DNA concentration in agarose inserts should not be greater than 80 micrograms/ml (10(7) diploid human cells/ml agarose insert).     

Orientation and stretching of DNA chains during pulsed field gel electrophoresis

The quantitative analysis of the decay of birefringence accompanying gel electrophoresis leads to the characterization of two phenomena respectively attributed to alignment of the tube and to overstretching of the chain in its tube. The major contribution to the molecular orientation is given by the overstretching which relaxes according to a stretched exponential law. The other process, slow, is characterized by a reptation time and a mean orientation factor in good agreement with the biased reptation model without overstretching.     

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.     

An electrophoretic karyotype for Schizosaccharomyces pombe by pulsed field gel electrophoresis.

The three chromosomal DNAs of S. pombe have been fractionated by pulsed field gel electrophoresis. The resulting molecular karyotype will greatly speed gene mapping in this organism, and it indicates that the separation range of the technique extends to DNA molecules as large as 9,000,000 base pairs.     

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.     

Atypical sieving of open circular DNA during pulsed field agarose gel electrophoresis

Pulsed field agarose gel (PFG) electrophoresis, originally used to improve the resolution by length of linear DNA [Cantor et al. (1988) Annu. Rev. Biophys. Biophys. Chem. 17, 287-304], is found here to cause atypical sieving of 48.5-97.0-kb open circular DNA. Two procedures of PFG electrophoresis are used: rotating gel electrophoresis with rotation of 2 pi radians [2 pi RGE; Serwer, P., & Hayes, S.J. (1989) Appl. Theor. Electrophor. (in press)] and field inversion gel electrophoresis [FIGE; Carle, G.F., Frank, M., & Olson, M. V. (1986) Science 232, 65-68]. During 2 pi RGE at 6 V/cm, the electrophoretic mobility (mu) of 48.5-kb open circular DNA increases in magnitude as agarose percentage (A) increases from 0.4 to 1.5. The sieving revealed by this mu vs A relationship is highly atypical (possibly unique) for any particle. The extent of this atypical sieving increases as electrical potential gradient, DNA length, and pulse time increase. In some cases a maximum is observed in a plot of mu's magnitude vs A. The mu of open circular lambda DNA is smaller in magnitude than the mu of equally long linear lambda DNA. Atypical sieving has also been observed by use of FIGE. As pulse times used during FIGE decrease below those achievable by 2 pi RGE, the progressive loss of circular DNA's atypical sieving is accompanied by both a dramatic increase in mu's magnitude at the lower A values and a decrease in mu's magnitude at the higher A values. At the lower A values, open circular DNA sometimes migrates more rapidly than linear DNA of the same length.(ABSTRACT TRUNCATED AT 250 WORDS)     

Techniques and high resolution DNA size markers for pulsed field gel electrophoresis

High resolution DNA size markers are described for pulsed field gel electrophoresis (PFGE). These markers provide resolution of 10–20 kbp over a size range from 10 kbp to more than 400 kbp and are produced by partial restriction digestion of lambda phage DNA concatemers (λ ladder). High resolution markers extending to over 400 kbp are made by partial restriction digestion of λ ladder embedded in agarose. Detailed methods are described for marker production and for DNA separation by contour-clamped homogeneous electric field (CHEF) electrophoresis. These markers and methods are useful for a variety of high resolution DNA mapping by PFGE.     

Genotypic characterization of Ureaplasma species by pulsed field gel electrophoresis

We describe the first use of pulsed field gel electrophoresis to genotype human Ureaplasma species. This technique can distinguish between U. urealyticum and U. parvum, differentiate most of the 14 serovars from one another, and identify differences among clinical isolates of the same serovar.     

Genotypic characterization of Ureaplasma species by pulsed field gel electrophoresis

We describe the first use of pulsed field gel electrophoresis to genotype human Ureaplasma species. This technique can distinguish between U. urealyticum and U. parvum, differentiate most of the 14 serovars from one another, and identify differences among clinical isolates of the same serovar.     

Rearrangements of yeast chromosomes revealed by pulsed field gel electrophoresis

Electrophoretic karyotypes of yeast Saccharomyces cerevisiae integrant strains containing the pYF91 plasmid integrated into the chromosomes I, III, VI, IX, XI were studied. A possibility was demonstrated of visual identification of the chimaeric chromosomes via the molecular weight increase by 13200 bp (the plasmid size) determined by pulsed field gel electrophoresis. Several gamma-rays induced rearrangements of the yeast chimaeric chromosome I causing instability in hybrids were also studied. The deletions induced in the I chromosome were analysed and their size estimated. The technique of pulsed field gel electrophoresis is recommended for determination of insertions and deletions in the chromosomes of yeast Saccharomyces cerevisiae.