Fluorometric determination of DNA in fixed tissue using ethidium bromide

The measurement of DNA in tissue samples fixed in ethanol/acetic acid is described. Small, fixed tissue samples are digested by warm alkaline treatment followed by neutralization with HCl, and DNA is determined by complex formation with the dye ethidium bromide (EB). When standard DNA from calf thymus was treated similarly, a hyperchromicity of 8–12% and a reduction in fluorescence intensity of the EB-DNA complex to 55% was observed. The NaOH concentration (0.5–2.0 mol/liter) or the temperature (50–60°C) used for the digestion of tissue, as well as subsequent ribonuclease or protease treatment had no effect on the observed tissue DNA concentrations.     

Fluorometric determination of ethidium bromide efflux kinetics in Escherichia coli

Background  

Efflux pump activity has been associated with multidrug resistance phenotypes in bacteria, compromising the effectiveness of antimicrobial therapy. The development of methods for the early detection and quantification of drug transport across the bacterial cell wall is a tool essential to understand and overcome this type of drug resistance mechanism. This approach was developed to study the transport of the efflux pump substrate ethidium bromide (EtBr) across the cell envelope of Escherichia coli K-12 and derivatives, differing in the expression of their efflux systems.     

Fluorometric methods employing low concentrations of ethidium bromide for DNA topoisomerase and endonuclease assays

DNA topoisomerase activity can be rapidly assayed by measuring the change in ethidium bromide fluorescence intensity after treatment of closed duplex DNA with enzyme. The sensitivity of the fluorometric assay has been enhanced 3-fold by a 10-fold reduction in ethidium bromide concentration to 0.1 microgram/ml. The results of the fluorometric assays are in close agreement with agarose gel electrophoretic analyses of reacted DNA. A sensitive fluorometric method using 0.1 microgram/ml ethidium bromide has also been developed to determine the fraction of nicked and linear DNAs in a mixture containing closed duplex DNA by measuring the fluorescence intensities of ethidium-DNA complexes at pH 7.0 and pH 12.0. These methods make possible very rapid and sensitive measurements of DNA topoisomerase and endonuclease activities.     

Measurements of DNA and RNA in mammary gland homogenates by the ethidium bromide technique.

A rapid method of determining simultaneously DNA and RNA in mammary gland homogenates using the ethidium bromide technique is discussed. The method utilizes a quantitative extraction of DNA and RNA with 2.0m sodium chloride, SDS, and EDTA at pH 8.0. Assays of mammary gland RNA and DNA using previously published methods were compared with determinations using the ethidium bromide technique. While the fluorescence method gave lower values for RNA when compared to those obtained using the orcinol or absorbancy ratio (OD 260nm/280nm), DNA measurements agreed well with the values determined by the diphenylamine technique. Extinction coefficient data for total mammary gland RNA isolated using a modified phenol extraction procedure are also presented.     

Mechanism of ethidium bromide inhibition of RNA polymerase

The effect of ethidium bromide on various steps of the reaction catalyzed by Escherichia coli DNA-dependent RNA polymerase is studied. Inhibition caused by low levels (approx. 6 μm) of this DNA-binding drug is a consequence of reducing the rate of RNA chain initiation; the rate of RNA chain growth is unaffected at this concentration. The sensitive step in the initiation process is the formation of stable complexes between RNA polymerase and initiation sites on the DNA. At higher levels (25 μm), ethidium bromide does inhibit the polymerization of those RNA molecules whose initiation has not been blocked.     

Connection of ethidium bromide with single-stranded DNA

The interaction of ethidium bromide with single-stranded synthetic and natural polynucleotides at high temperatures (t = 70 degrees C) and low pH values (pH 3.0) was studied. The isotherms of adsorption of ethidium bromide on single-stranded DNA were obtained. Two modes of binding of single-stranded DNA, strong and weak, were revealed. The values of the corresponding constants of interaction of this ligand and the number of bases per one binding site were determined.     

Renaturation of DNA in the presence of ethidium bromide

The rate of renaturation of T2 DNA has been studied as a fuction of ethidium bound per nucleotide of denatured DNA. The Binding constants and number of binding sites for ethidium have been determined by spectral titration for denatured DNA at 55, 65, and 75°C and for native DNA at 65°C in 0.4M Na⁺. The rate of renaturation of T2 DNA was found to be independentof ethidium binding up to 0.03 moles per mole of nucleotide. Above 0.03 moles, the rate drops off precipitously approaching zero at 0.08 and 0.06 moles bound ethidium per nucleotide at 65°C respectively. A study was also made of the use of bound ethidium fluorescence as a probe for monitoring DNA renaturation reactions.     

Mechanism of ethidium bromide inhibition of RNA polymerase.

2-Oxo-3-phenyl-1,3-oxazetidine was found to thermally undergo a 2,2-cyclo-reversion reaction with an enthalpy of activation of 30.8 Kcal. This suggests that an oxazetidine fused to a flavin could be the labile light producing intermediate in the bacterial luciferase reaction since the reaction would be favored by ring fusion and the lower excited state of flavin.     

Protocol for high-sensitivity/long linear-range spectrofluorimetric DNA quantification using ethidium bromide

Ethidium bromide (EtBr) is the most widely used fluorescent dye in nucleic acid gel electrophoresis since decades. However it has been essentially forgotten in DNA quantification by spectrofluorimetry. While investigating sensitivity and dynamic range of available fluorochromes, we found that EtBr permits much more sensitive fluorimetric measurements than previously thought. We report here a revised, accurate, and easy-to-use protocol for EtBr-based DNA quantification in solution, which usefully complements the widely used indirect quantification on agarose gels.     

Spectrophotometric analysis of RNA and DNA using cetyltrimethylammonium bromide

A versatile procedure is described for the analysis of RNA and DNA in brain using cetyltrimethylammonium bromide as the initial precipitant. Optimal conditions are described for the precipitation, hydrolysis, and effective separation of the RNA and DNA fractions from contaminating protein. The RNA and DNA fraction can now be accurately estimated by uv absorbance without a two wavelength correction. This method has also been used for the analysis of other mammalian organs and for mammalian cells obtained from tissue culture. The method may also be used for the simultaneous determination of radioactivity in nucleic acids. The orcinol reaction is shown to give high values for brain RNA.     

Improvements in the ethidium bromide method for direct fluorometric estimation of DNA and RNA in cell and tissue homogenates

In the DNA and RNA assays carried out with the use of ethidium bromide in cell and tissue homogenates according to the method of Karsten and Wollenberger (1), Pronase can be replaced to advantage by heparin in the nucleoprotein dissociation step. Furthermore, rhodamine B is used as a standard instead of DNA. Attention is called to several methodical details.     

Sensitive spectrophotometric determination of tetraphenylboron and tetraphenylarsonium with ethidium bromide.

Tetraphenylboron and tetraphenylarsonium ions have been determined spectrophotometrically by measuring the red shift in the absorbance maximum of ethidium on its reaction with tetraphenylboron at neutral pH. The present method permits the determination of nanomole quantities of these ions.