Simultaneous electroelution and concentration of DNA fragments from agarose gels

A rapid and inexpensive method for the electroelution of DNA fragments from agarose gels is described. DNA fragments were separated by agarose gel electrophoresis and visualized by staining with ethidium bromide. Selected DNA fragments were placed into electroeluter tubes capped with dialysis membrane and electroeluted into a small volume of buffer using a conventional horizontal gel electrophoresis apparatus. The method successfully eluted and concentrated DNA fragments with molecular weights ranging from 2.7 to 13.9 MDa in 3 h.     

A rapid method for diagnosis of the Lebanese allele in the low-density lipoprotein receptor gene.

The Lebanese allele in the low-density lipoprotein receptor gene is one of the alleles which results in the disease familial hypercholesterolemia. We describe a rapid method for detection of the Lebanese allele, using the polymerase chain reaction to amplify part of exon 13, intron 14 and all of exon 14. The amplified DNA is then digested with HinfI which distinguishes between the normal and Lebanese alleles. A previously unidentified HinfI site is described in the intron. HinfI fragments are separated using polyacrylamide gel electrophoresis, and visualized by ethidium bromide staining.     

Comparison of ethidium bromide and 4′,6-diamidino-2-phenylindole as quantitative fluorescent stains for DNA in agarose gels

Two methods of quantitative, fluorescent detection of DNA bands in agarose gels separated by electrophoresis were evaluated for sensitivity and linearity of response. Comparisons of ethidium bromide staining with a method using 4′,6-diamidino-2-phenylindole (DAPI) developed in this work showed that DAPI is several times more sensitive than ethidium bromide in conditions of comparable background flourescence. Optimum flourescent staining and detection conditions for DNA bands in agarose gels using DAPI are desctribed, and advantages of this method over other fluorescent detection methods are discussed.     

Ethidium bromide resistance in a rat liver epithelial cell line: Association with enhanced drug efflux

Ethidium bromide-resistant cell strains were obtained by continuous selection of an adult rat liver-derived cell line (ARL6T) grown in the continuous presence of 200 ngl ml ethidium bromide. Comparison of resistant strains and parental (sensitive) cells was made for uptake and binding of ethidium bromide, visualized as fluorescent ethidium bromide-nucleic acid complexes. Although uptake of ethidium bromide was similar in parental and resistant cells, efflux kinetics were markedly different. Over a three-hour period, parental (sensitive) cells maintained fluorescence following a short ethidium bromide pulse (100 g/ ml ethidium bromide). In contrast, ethidium bromide-resistant cell lines eliminated photographically detectable fluorescent complexes within three hours following pulse exposure to ethidium bromide. The rapid elimination of ethidium bromide fluorescent complexes in all (5) resistant cell strains examined supports an efflux mechanism as contributing to the resistance of ethidium bromide cytotoxicity in these cells.Abbreviations EtBr ethidium bromide - HBSS Hanks' balanced salt solution     

Simple method for demonstrating small plasmid deoxyribonucleic acid molecules in oral streptococci.

A simple procedure for rapidly demonstrating small plasmids (less than 10 megadaltons) in oral streptococci is described. Logarithmic-phase, glycine-treated cells from 1.5-ml broth cultures were converted to osmotically fragile forms and lysed with sodium dodecyl sulfate. After the hydrodynamic shearing of host chromosomal deoxyribonucleic acid, such lysates were analyzed by low-voltage agarose gel electrophoresis. Small plasmids, migrating significantly faster than chromosomal deoxyribonucleic acid under such conditions, were readily visualized by ethidium bromide staining.     

Cytological Analysis of Anastomoses and Vegetative Incompatibility Reactions in <Emphasis Type="Italic">Helicobasidium monpa</Emphasis>

Our examination of the cytological characteristics of the vegetative incompatibility reaction in a filamentous basidiomycete, Helicobasidium monpa, by analyzing the fluorescence emitted by ethidium bromide and acridine orange stained nuclei is described. Hyphal anastomoses between strains belonging to different mycelium compatibility groups (MCG) were observed with cell death in fused hyphae, whose nuclei were intensified by ethidium bromide. In contrast, the nuclei in a living cell were not intensified by staining with ethidium bromide, but were intensified by staining with acridine orange. These results indicate that in H. monpa, ethidium bromide staining is a useful method for detecting dead cells. We also examined the relationships between the alternation of ploidy and hyphal anastomosis formation using the newly developed method on filamentous fungi. The tetraploid monokaryon strain derived from the original dikaryon strain by continuous subculture could not be fused to any wild type strains, but the original dikaryon strain could be fused without cell death to only the same MCG strain. In contrast, the haploid dikaryon strain derived from the original monokaryon strain fuses to several strains belonging to different MCGs without cell death. These results suggested that the cellular ploidy of this fungus is closely related to its mating system and, H. monpa may be a self-fertilizing fungus. Received: 13 June 2001 / Accepted: 8 August 2001     

Different effects of 33258 Hoechst and DAPI in fluorescent staining of sister chromatids differentially substituted with bromodeoxyuridine

Summary After substitution with 5-bromodeoxyuridine (BrdUrd) for two rounds of replication, chromosomes in cytological preparations stained with 33258 Hoechst show upon epiluminescence an immediate differential sister chromatid fluorescence. When stained with DAPI, however, which has a structural resemblance to part of the 33258 Hoechst molecule, such a differential pattern of fluorescence was only induced after some delay. Upon restaining with the same dye the differential fluorescence appeared instantly. In preparations double stained with ethidium bromide and 33258 Hoechst the induction of a differential staining of sister chromatids with 33258 Hoechst was not accompanied by a differential staining with ethidium bromide. Once a differential staining was obtained with DAPI in preparations double stained with ethidium bromide and DAPI, the ethidium bromide pattern also appeared to be differential upon subsequent observation. No differentiation could be obtained with ethidium bromide alone. The observations described in the case of 33258 Hoechst staining are in agreement with a molecular quenching by BrdUrd without gross structural consequences for the DNA. In the case of DAPI staining, however, there occurs a differential photolysis of BrdUrd-substituted DNA. Besides the nature, most likely the size, of the fluorochrome molecules themselves, the state of the fixed chromatin appeared also to play a role in determining the mechanism of the sister chromatid differentiation: after prolonged incubation in buffer, BrdUrd-containing chromosomes stained with 33258 Hoechst showed a differential staining evidently caused by photolysis, indicating that they had become more susceptible to light.     

Use of ethidium for the cytochemical study of chromatin]

A cytochemical method of chromatin study by means of nuclear staining with ethidium (bromide) is described. Dependence of stain binding by chromatin on ethidium concentration, ionic composition and buffer pH value has been analyzed. It is suggested that cells be stained in 2.10(-5) M solution of ethidium in 0.1 M tris-HCl buffer at pH 8.0 during 30 min. The fluorescence of nuclei stained with ethidium under conditions described is shown to reflect changes in physico-chemical properties of chromatin taking place in the course of its chemical modification and physiological activation in regenerating liver. The use of ethidium for chromatin cytochemistry allows to study chromatin properties in wide ranges of pH. Some other advantages of the method suggested over the commonly used method of acridine orange staining are discussed.     

Fluorescence of yeast vitally stained with ethidium bromide and propidium iodide

Both ethidium bromide and propidium iodide stain growing yeast. As visualized in the fluorescence microscope, ethidium stains the nucleus and cytoplasm in wild type yeast and in those grown in 10% dextrose, with brightly fluorescent cytoplasmic granules being present in both. Under the latter conditions, the mitochondria are repressed but not absent. In rho 0 cells, in which the mitochondrial DNA is absent, ethidium appears to bind to the cell wall or membrane preferentially with no cytoplasmic granules being visible. In all cell types, propidium appears to bind the cell wall or membrane with no cytoplasmic granules being visible in any cell. The staining patterns thus suggest greater differences in the binding of these two types to mitochondrial DNA in situ than is suggested by their in vitro behavior. These differences in binding could explain their different mutagenic capacities..     

Cloning of RNA molecules in vitro.

A method for RNA amplification in an immobilized medium is described. The medium contains a complete set of nucleotide substrates and purified Q beta replicase, an enzyme capable of exponentially amplifying RNAs under isothermal conditions. RNA amplification in the immobilized medium results in the formation of separate 'colonies', each comprising the progeny of a single RNA molecule (a clone). The colonies were visualized by staining with ethidium bromide, by utilizing radioactive substrates, and by hybridization with sequence-specific labeled probes. The number and identity of the RNA colonies corresponded to that of the RNAs seeded. When a mixture of different RNA species was seeded, these species were found in different colonies. Possible implementations of this technique include a search for recombinant RNAs, very sensitive nucleic acid diagnostics, and gene cloning in vitro.     

Imaging and measuring the rituximab-induced changes of mechanical properties in B-lymphoma cells using atomic force microscopy

The topography and mechanical properties of single B-lymphoma cells have been investigated by atomic force microscopy (AFM). With the assistance of microfabricated patterned pillars, the surface topography and ultrastructure of single living B-lymphoma cell were visualized by AFM. The apoptosis of B-lymphoma cells induced by rituximab alone was observed by acridine orange/ethidium bromide (AO/EB) double fluorescent staining. The rituximab-induced changes of mechanical properties in B-lymphoma cells were measured dynamically and the results showed that B-lymphoma cells became dramatically softer after incubation with rituximab. These results can improve our understanding of rituximab’effect and will facilitate the further investigation of the underlying mechanisms.     

Spot blot: a hybridization assay for specific DNA sequences in multiple samples

Small aliquots of DNA-containing samples from gradient or column fractions are spotted onto agarose plates containing ethidium bromide, and the DNA is visualized qualitatively. After it is overlaid with a thin agarose cover, the DNA is denatured and transferred to nitrocellulose filters for hybridization. This method, called the "spot-blot assay," is particularly convenient when dealing with large numbers of samples.     

Silver staining of native and denatured eucaryotic DNA in agarose gels

A modified method of silver staining for native and denatured eucaryotic DNA in 1% agarose gel is described. This method is at least fivefold more sensitive than ethidium bromide staining, with a detection limit of 2.5 ng for total DNA. The calibration curve is linear within the range 5-30 ng of single-stranded and double-stranded DNA. This method is especially advantageous for electrophoretic assessment of DNA molecular weights.     

Faster,safer, and better DNA purification by ultracentrifugation using GelRed stain and development of mismatch oligo DNA for genome walking

Purification of plant DNA involves lengthy ultracentrifugation using ethidium bromide. Here, ultracentrifugation method is improved by staining with GelRed. The resulting method is faster, safer and of higher sensitivity. Purified DNA quality was confirmed by treatment with restriction enzymes and isolation of gene promoters. New type of long adaptor with mismatch sequence was also developed for promoter isolation.     

Histochemical applications of two phenanthridinium compounds

The fluorescent compounds ethidium monoazide and ethidium bromide were found to react intensely with nucleic acids of fixed, paraffin embedded tissues of rat and mouse. For routine staining, 10(-5) M solutions of ethidium bromide and its monoazide analogue were virtually identical in their reactions. Fresh frozen sections of the tissues reacted in the same manner as fixed, paraffin embedded samples. Fluorescence of DNA and RNA in rat pancreas could be selectively abolished by taking advantage of the greater sensitivity of RNA to acid hydrolysis. Hydrolysis in aqueous solutions (1 N HCl at 55-60 C) abolished RNA fluorescence in 5 min, whereas 20 min or longer were required to destroy DNA fluorescence. DNA fluorescence was selectively abolished by 3 hr in 0.1 N HCl in anhydrous methanol while the RNA remained unaffected. Rat pancreas stained with the 10(-5) M ethidium compounds below pH 5.0 showed reduced RNA fluorescence, but the DNA continued to fluoresce brightly at pH 0.6. Reducing the pH of the staining solution to pH 1.0, therefore, was an additional method of selectively abolishing RNA fluorescence. Ethidium solutions in 5.0 M NaCl at pH 5.0 had little effect on DNA or RNA fluorescence. This new method of examining nucleic acids in fixed tissue samples opens new approaches to the histochemistry of these substances. The method also offers new possibilities for the study of mutagenic drug-DNA interactions.     

Histochemical Applications of Two Phenanthridinium Compounds

The fluorescent compounds ethidium monoazide and ethidium bromide were found to react intensely with nucleic acids of fixed, paraffin embedded tissues of rat and mouse. For routine staining, 10^-5 M solutions of ethidium bromide and its monoazide analogue were virtually identical in their reactions. Fresh frozen sections of the tissues reacted in the same manner as fixed, paraffin embedded samples. Fluorescence of DNA and RNA in rat pancreas could be selectively abolished by taking advantage of the greater sensitivity of RNA to acid hydrolysis. Hydrolysis in aqueous solutions (1 N HCl at 55-60 C) abolished RNA fluorescence in 5 min, whereas 20 min or longer were required to destroy DNA fluorescence. DNA fluorescence was selectively abolished by 3 hr in 0.1 N HCl in anhydrous methanol while the RNA remained unaffected. Rat pancreas stained with the 10^-5 M ethidium compounds below pH 5.0 showed reduced RNA fluorescence, but the DNA continued to fluoresce brightly at pH 0.6. Reducing the pH of the staining solution to pH 1.0, therefore, was an additional method of selectively abolishing RNA fluorescence. Ethidium solutions in 5.0 M NaCl at pH 5.0 had little effect on DNA or RNA fluorescence. This new method of examining nucleic acids in fixed tissue samples opens new approaches to the histochemistry of these substances. The method also offers new possibilities for the study of mutagenic drug-DNA interactions.     

Subcellular localization of photoreactive ethidium analogs in Trypanosoma brucei by fluorescence microscopy

To identify the in vivo targets of the trypanocide, ethidium bromide, the fluorescent staining of T. brucei was examined for a series of ethidium analogs using fluorescence microscopy. Determination of the biological targets for most drugs is limited by the reversible nature of their interactions. To overcome this limitation, photoaffinity (azido) analogs of ethidium, which are capable of covalent attachment with photoactivation, were used to identify the ethidium binding sites within the parasites. Two of these compounds, when covalently attached, demonstrated an enhancement of fluorescent staining and were selective for the kinetoplast at low drug concentrations. These compounds were also those found previously to have the highest trypanocidal activity. Propidium, a phenanthridinium analog identical to ethidium except for a larger, more ionic substitution at R5, showed more nonspecific binding as determined by its general staining of the cytoplasm.     

Highly Selective Acridine and Ethidium Staining of Bacterial DNA and RNA

The acridine dyes acridine orange (AO) and coriphosphine O (CPO) and ethidium bromide (EtBr) were used to stain bacterial digests after electrophoresis in native and denaturing (SDS) polyacrylamide gels and were shown to stain DNA and RNA preferentially over other subcellular components in the gels. Vegetative cell digests of Bacillus subtilis, Escherichia coli, Micrococcus luteus, and Staphylococcus aureus showed intense staining of DNA with AO and CPO near the top of the gel, but little or no staining of other cellular constituents. EtBr stained both DNA and RNA in the gels. Protein standards and non-nucleic acid cellular constituents stained faintly with high concentrations (> 100 μM) of AO, lower concentrations (13.9 μM) of CPO, and did not stain with 0.5 μg/ml EtBr in denaturing gels. The complete set of cellular biochemicals was visualized by silver staining, while the protein subset was detected by Coomassie blue staining. The highest concentrations of AO (120 μM) and CPO (13.9 μM) were shown to detect purified DNA in gels with a sensitivity in the range of 25–50 ng per band. This work demonstrates the specificity of acridine and ethidium dyes for nucleic acids, while illustrating the level of non-nucleic acid-specific interactions with other cellular components by staining of electrophoretically separated cellular components in a gel matrix.     

Randomly amplified polymorphic DNA (RAPD) for rapid typing of Lactobacillus plantarum strains

Randomly amplified polymorphic DNA (RAPD) has been used for rapid typing of Lactobacillus plantarum strains. RAPD was used with either purified chromosomal DNA serving as template in the polymerase chain reaction, or with crude cell extracts, and using a 9-mer primer with 80% G + C content. Amplified DNA was visualized by ethidium bromide staining after separation on agarose gels. Patterns from 20 Lact. plantarum strains and two Lact. pentosus strains were analysed using the Pearson products moment correlation coefficient ( r ) and the unweighted pair group method with arithmetic averages (UPGMA). With some exceptions, the two sources of template DNA gave the same clusters and subclusters of strains at the similarity level of 50%. About 50% of the strains could be individually separated from all the other tested strains. The buffer brand, the amount of primer and crude cell extract used in the PCR-step were crucial for the final pattern.