Separation of chromosomal DNA molecules from yeast by orthogonal-field-alternation gel electrophoresis

A simple agarose-gel apparatus has been developed that allows the separation of DNA molecules in the size range from 50 kb to well over 750 kb, the largest size for which size standards were available. The apparatus is based on the recent discovery that large DNA molecules are readily fractionated on agarose gels if they are alternately subjected to two approximately orthogonal electric fields. The switching time, which was on the order of 20-50 sec in our experiments, can be adjusted to optimize fractionation in a given size range. The resolution of the technique is sufficient to allow the fractionation of a sample of self-ligated lambda DNA into a ladder of approximately 15 bands, spaced at 50 kb intervals. We have applied the technique to the fractionation of yeast DNA into 11 distinct bands, several of which have been shown by DNA-DNA hybridization to hybridize uniquely to different chromosome-specific hybridization probes. In this paper, we describe the design of the apparatus, the electrophoretic protocol, and the sample-handling procedures that we have employed.     

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.     

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.     

Rapid microsatellite analysis using discontinuous polyacrylamide gel electrophoresis.

A method for screening large numbers of samples for microsatellites using discontinuous, non-denaturing polyacrylamide gels and rapid fluorescent gel staining is described. Disc electrophoresis on slab gels provides high-resolution of PCR products. It is useful for collecting population data once microsatellite loci have been characterized.     

Analysis of tubulin isoforms by two-dimensional gel electrophoresis using SDS-polyacrylamide gel electrophoresis in the first dimension.

A two-dimensional electrophoretic system using SDS-polyacrylamide gel electrophoresis (SDS-PAGE) in the first dimension and isoelectric focusing (IEF) in the second dimension was devised. In spite of its simplicity, this method could show a markedly high resolution for tubulin isoforms and moreover could classify them into alpha- or beta-tubulin as a two-dimensional profile. With this method, seven alpha- and four beta-tubulin isoforms could be detected within axoneme from Tetrahymena cilia. Moreover this method could also resolve tubulin isoforms from the rabbit brain. These results indicate that the present two-dimensional gel electrophoresis is a useful tool for the electrophoretic analysis of tubulin isoforms from various sources.     

Reovirus-specific polypeptides: analysis using discontinuous gel electrophoresis.

The electrophoretic analysis of reovirus-specific polypeptides in infected cells using a discontinuous gel system has allowed the resolution of additional viral-specific polypeptides, including one large-sized gamma3 and two (or possibly three) medium-sized (mu3, mu4, mu5(?)) species. The proteins designated mu0, sigma1, and sigma2 based on electrophoretic mobility in gel systems containing phosphate-urea correspond to mu4, sigma2, and sigma1, respectively, when analyzed in systems containing Tris-glycine. It is likely that protein modifications (phosphorylation and glycosylation) are responsible for at least some of these differences.     

Analysis of heat-denatured DNA using native agarose gel electrophoresis.

The use of native or neutral gels to resolve denatured DNA affords a rapid and convenient analytical method for assessing the consequences of a number of procedures employed in molecular biology research. We demonstrate that this method can be used to analyze transition melting temperature (Tm) and strand breakage in heat-denatured duplex DNA. This shows that some commonly recommended denaturation procedures can result in significant degradation of DNA and that reannealing or aggregation can occur when samples are concentrated or ionic conditions altered.     

Detection of deoxyribonucleoside-triphosphate imbalance death-induced DNA double strand breakage in FM3A cells by orthogonal-field-alternation gel electrophoresis (OFAGE)

The mechanism of intracellular deoxyribonucleoside-triphosphate (dNTP) imbalance death of mouse mammary tumor FM3A cells was studied. When the cells were exposed to 5-fluorodeoxyuridine, deoxyadenosine, or 2-chlorodeoxyadenosine, dNTP pool imbalance resulted. The imbalance was followed by DNA double strand breaks and subsequent cell death. The DNA double strand breaks have been directly examined by means of orthogonal-field-alternation gel electrophoresis (OFAGE). Fragmented DNA band appeared to be approximately 100-200 kb in size.     

Analysis of the fibrin-polymerizing reaction using sodium dodecylsulfate-agarose gel electrophoresis.

1. A new method of sodium dodecylsulfate gel electrophoresis, sodium dodecyl-sulfate-agarose gel electrophoresis, was developed. The new electrophoresis was shown to have a high and efficient resolving power for fibrin polymers and was also applicable to other proteins. 2. By sodium dodecylsulfate-agarose gel electrophoresis, the fibrin polymerizing reaction with activated fibrin stabilizing factor was analyzed in detail. The cross-linking between the gamma-chains was indicated to occur at an intermolecular level. The solubility of polymerized fibrin was suggested to be related mainly to the formation of the crosslinks between gamma-chains.     

Assay of proteoglycan populations using agarose-polyacrylamide gel electrophoresis

The agarose-polyacrylamide gel electrophoresis procedure for the analysis of proteoglycans originally described by C. A. McDevitt and H. Muir (1971, Anal. Biochem. 44, 612-622) has been modified to minimize trailing and to allow the analysis of crude samples, i.e., tissue extracts. A slab gel system was used, permitting reproducible analysis of many samples. Procedures are described that can be used to separate and quantify several subpopulations of proteoglycans and also to quantify the proportion of proteoglycans capable of aggregating with hyaluronic acid. Applications of the procedure include transfer to nitrocellulose paper followed by immunological detection of proteoglycans as well as fluorography of separated, radiolabeled proteoglycans.     

SDS-polyacrylamide gel electrophoresis of lipopolysaccharides.

Lipopolysaccharides (LPS) prepared from four different species of Neisseria have been separated by SDS-polyacrylamide gel electrophoresis. Each LPS possessed a characteristic mobility on gels. Examination of the effect of acrylamide concentration on migration illustrated that the basis of the separation was molecular size and not intrinsic charge.     

Protein fragment domains identified using 2D gel electrophoresis/MALDI-TOF.

We previously reported a protein expression profiling experiment conducted on human pancreatic tissues using 2D gel electrophoresis and mass spectrometry. Here, 18 spots that were identified in the gel at molecular weights more than 10 kDa lower than database values are characterized. The matrix-assisted laser desorption/ionization mass spectrometry coverage is sufficient to identify the protein region present in each spot. Most of the fragments correspond to processed chains and known structural or functional domains, which may result from limited proteolysis.     

Studies of dynein from Tetrahymena cilia using agarose polyacrylamide gel electrophoresis.

Described in this report is an application of agarose-polyacrylamide gel electrophoresis, which separates protein components of crude dynein fraction (Fraction I by Gibbons) derived from Tetrahymena cilia. By this method, the fraction was separated into three protein components (designated as bands I, II and III) on the gel. When the gel was actively stained for dynein ATPase, a single band appeared, which coincided with the position of band I. A purified dynein prepared by controlled pore glass (CPG-10) column chromatography and followed by Biogel A-15m filtration showed one band on the gel at the same position as band I. These results suggest that among these three protein components, band I represents dynein and bands II and III are derived form non-ATPase protein. 'Burstic phenomenon' was also observed on their ATPase activity when axoneme or crude dynein fractions were used for ATPase assay, while the phenomenon was almost extinguished when partially purified dynein after controlled pore glass column chromatography was used as sample.