Quantitative fluorescence of DNA-intercalated ethidium bromide on agarose gels

Techniques for analyzing DNA distributions on agarose gels are examined by both two-dimensional and one-dimensional methods. It is demonstrated that very large errors in DNA concentration occur in such analyses unless (i) the electrophoresis is performed in a careful, reproducible manner, (ii) the films are calibrated with an internal standard, (iii) high resolution densitometry is used for analyzing the films, and (iv) appropriate background controls are used to determine the baselines for integration. Two-dimensional scanning produces more accurate results than one-dimensional scanning, but in cases where the bands are relatively uniform, the one-dimensional analysis gives good results. A technique for determining accurate distributions is described.     

An assay for proteinases and their inhibitors based on DNA/ethidium bromide fluorescence

Proteinases and their inhibitors have become the subject of intense research interest recently, since they control a multitude of very important biological processes, from the development of lambda phage to hypertension in humans. We have developed a simple and sensitive assay for detecting the activity of proteinases and of their proteinase inhibitors. The assay is based on ethidium bromide fluorescence, according to the following principles: (i) Ethidium bromide increases its fluorescence by 25-fold when it intercalates between base pairs of double-stranded DNA. (ii) Histones prevent this large increase in fluorescence by binding with high affinity to DNA thus blocking ethidium bromide intercalation. (iii) A proteinase that digests histones will make more DNA available for ethidium bromide intercalation, thereby producing an increase of fluorescence. Proteinase activity can easily be determined, in the presence of a DNA/histone complex, from the rate of ethidium fluorescence increase. In contrast, activity of a proteinase inhibitor is quantitated by the inhibition of fluorescence gain in the presence of a known amount of proteinase. This assay is rapid, simple, inexpensive, and, at the same time, accurate and sensitive enough to allow quantitation of nanogram amounts of various broad-specificity proteinases and their inhibitors. We show some possible applications of the assay (i) in testing column fractions during protein purifications, (ii) quantitation of alpha 1-antitrypsin in human serum, and (iii) detection of proteinase activity in cell extracts.     

A synthesis of labeled ethidium bromide

A method is described for the synthesis of labeled ethidium (3,8-diamino-5-ethyl-6-phenyl phenanthridinium) bromide. Based on benzoic acid, the radioactive precursor used, the yield is 15% of a compound that is indistinguishable from authentic ethidium bromide in its absorption spectra (uv, visible, ir), Chromatographie behavior, and mutagenic effectiveness in the induction of respiration-deficient cell lines in baker's yeast.     

A simple and sensitive method for the quantitative estimation of collagen.

A method to quantitate collagen in solution is described. It is based on binding of the dye Sirius Supra red F3BA by collagen followed by elution and estimation of the bound dye in a spectrophotometer. The assay can be used in the range from 10 to 100 μg protein. The method is rapid, specific, simple, sensitive, and highly reproducible.     

Environment-induced changes in DNA conformation as probed by ethidium bromide fluorescence

The interaction of the ethidium cation with calf thymus DNA is investigated in solutions of different ionic strength and temperature by observation of the enhancement of fluorescence of ethidium upon intercalation in the duplex structure. The quantum yield of the fluorescence of the intercalated dye is found to increase either upon lowering the Na+ concentration or upon increasing the temperature. The existence of a correlation between the geometry of the intercalation complex and the features of the secondary structure of DNA is suggested. Binding isotherms under corresponding environmental conditions are also quantitated by fluorescence enhancement and interpreted in terms of the neighbor exclusion model. Large contributions from change in hydration to the thermodynamics of binding are demonstrated by the temperature dependences of the equilibrium constants. The neighbor exclusion range is found to be practically independent of the salt concentration but its value increases from an average of 2.4 around room temperature to 4-5 at 80 degrees C, as inferred from the binding curves in 0.15 and 0.5 M [Na+] or from the DNA hypochromism vs temperature profiles of complexes at 10(-3) M [Na+]. All the data point to a possible sequence-conformation specificity in the intercalation of ethidium which in heterogeneous DNA is mediated by environmental changes.     

A simple quantitative method for the estimation of free ecto-sulfhydryl groups of spermatozoa

A simple rapid quantitative method has been developed for the estimation of sperm ecto-SH groups on the basis of their high affinity binding to the mercurial: [203Hg]p-chloromercuriphenylsulfonic acid (PCMPS) used as a surface probe. The thiol reagent did not penetrate the sperm plasma membrane, as evidenced by the extremely rapid time course of the binding reaction and undetectable uptake of [203Hg]PCMPS by intact goat spermatozoa. The binding reaction was not due to contaminating broken or damaged cells, if any. The method consists of incubating of highly motile goat spermatozoa with PCMPS in a modified Ringer solution at 37 degrees C for 5 min, agglutination of the labelled cells with polyethyleneimine (100 micrograms/ml) and filtration and washing of the cell suspension through Whatman No. 1 filter discs under mild vacuum. The binding interaction is proportional to cell concentration, specific and saturable at 50 microM PCMPS. The method is capable of estimating free ecto-SH as low as 25 pmoles. Spermatozoa possess 286 +/- 61 pmoles of free ecto-SH groups/10(6) cells. Scatchard analysis showed the presence in goat spermatozoa of multiple classes of ecto-SH groups differing in their affinity for PCMPS.     

Terbium identifies double-stranded RNA on gels by quenching the fluorescence of intercalated ethidium bromide

We report that the lanthanide cation terbium quenches the fluorescence of ethidium bromide bound to double-stranded RNA by 40-fold, whereas the quenching of double-stranded and single-stranded DNA is under 2.5-fold and the quenching of single-stranded RNA is under 5-fold. This observation was used to develop a convenient method of detecting dsRNA among other nucleic acids in an agarose or polyacrylamide gel. The sensitivity of the method is approximately 4 ng/mm2.     

Parallel and antiparallel DNA: fluorescence quenching of ethidium bromide by 7-deazapurines

The fluorescence quenching of ethidium bromide caused by 7-deaza-2'-deoxyguanosine or 7-deaza2'-deoxyisoguanosine is observed in DNA with parallel or anti-parallel chain orientation.     

A yeast mitochondrial deoxyribonuclease stimulated by ethidium bromide

Several DNase activities, with different substrate and pH requirements, have been identified in yeast mitochondria. One of them is active on double stranded DNA at neutral pH and stimulated by Ethidium Bromide and other DNA intercalating drugs. This activity could be responsible for the yeast mitochondrial DNA degradation induced during mutagenesis by Ethidium Bromide.     

Solid-phase plate-reader quantification of specific PCR products by measurement of band-specific ethidium bromide fluorescence

Real-time PCR is widely employed to quantify PCR products across a range of applications. However, accurate real-time PCR is not always technically feasible, and alternative methods for PCR product quantification can be expensive and time consuming to validate. We have developed an inexpensive, rapid, and immediately accessible protocol to quantify PCR products, by measuring ethidium bromide fluorescence of PCR products excised from agarose gels. This protocol has relevance to a broad range of methods in molecular biology where quantification of PCR products is necessary.     

Determination of membrane-bound chloroplast RNA by the ethidium bromide fluorometric method

The method of El-Hamalawi et al. [(1975) Anal. Biochem.67, 384–391] for the fluorometric determination of nucleic acids with ethidium bromide has been adapted for the assay of membrane-associated chloroplast RNA. Membranes are stripped of RNA by incubation in a high-salt buffer lacking Mg²⁺, and the RNA is collected by magnesium phosphate-ethanol coprecipitation. RNA levels are determined by measuring the degree of enhancement of ethidium bromide fluorescence.     

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.