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Because superoxide is involved in various physiological processes, many efforts have been made to improve its accurate quantification. We optimized and validated a superoxide-specific and -sensitive detection method. The protocol is based on fluorescence detection of the superoxide-specific hydroethidine (HE) oxidation product, 2-hydroxyethidium. We established a method for the quantification of superoxide production in isolated mitochondria without the need for acetone extraction and purification chromatography as described in previous studies.

Various techniques for quantification of PCR are available. Most frequently, the densitometric intensities of ethidium bromide-stained PCR products separated in gels are compared after normalizing to the levels of housekeeping gene products such as beta-actin. More precise, but extremely time consuming, is the technique of competitive PCR. Newer methods, such as tracking amplification in real-time, have high start-up and maintenance costs (e.g., TaqMan, Applied Biosystems; LightCycler, Roche; I-Cycler, Bio-Rad). Here, I describe an alternative, simple technique to quantify PCR products by determining the entire phosphate released during PCR. The method can be performed using common laboratory equipment, and the reagents needed are extremely cheap. The method is validated by measuring the induction of inducible nitric oxide synthase gene expression in cell culture and comparing the results with data obtained by LightCycler experiments and RNase protection assays.

Most conjugate proteins undergo both conformational and stability changes on ligand removal. When architecture remains unchanged in the protein holo and apo forms, it is uncertain whether the protein stability also remains unaltered in both of the forms. Neocarzinostatin (NCS), a chromoprotein possessing a potent enediyne chromophore stands for such an instance. Protein–chromophore interaction has not been thoroughly explored previously due to a lack of strategies to independently and simultaneously monitor changes in the NCS conjugates. Here we report a method by which one can detect the signal exclusively from only one of the NCS conjugates without the spectral interference from the other. Stability of the NCS protein is significantly correlated to the protein-bound chromophore, irrespective of denaturation by heat, pH, urea, or ethanol. Despite the similarity in protein backbone conformation, protein stability of the NCS holo form diminishes and equalizes to that of the apo form when the chromophore is released and degraded. Although the enediyne chromophore is highly unstable, it intriguingly protects the protein by which it is protected. Significant mutual reliance between the carrier protein and its naturally associated ligand unveils important information on the NCS drug stability.

Quantitative studies of the binding of various DNA-binding antibiotics with dsDNA are useful for drug design, not only for effective antibiotics, but also for antitumor drugs. We studied the binding kinetics, association and dissociation rate constants, and association constants (kon, koff, and Ka, respectively) of intercalators and groove binders, including various antibiotics, to double-stranded DNA (dA30·dT30 and dG30·dC30) immobilized on a highly sensitive 27 MHz quartz-crystal microbalance (QCM) in aqueous solution. Although a simple ethidium bromide intercalator bound to both dA30·dT30 and dG30·dC30, antibiotics that are side-binding intercalators, such as daunomycin, aclacinomycin A, and actinomycin D, with sugar or peptide moieties on the intercalator parts selectively bound to dG30·dC30 with high Ka and small koff values. Nogalamycin, a dumbbell-shaped penetrating intercalator, showed low kon and koff values owing to slow duplex unwinding during the penetration process. Groove binders (Hoechst 33258, distamycin A, and mithramycin) had high Ka values owing to the high kon values. Kinetic parameters depended largely on molecular shapes and DNA-binding molecule binding modes.

We have developed a simple measuring system for fluorescence-detected linear dichroism and applied it to the structural analysis of the RecA-DNA complex filaments, which are intermediates of the homologous recombination reaction. Taking advantage of the selectivity of fluorescence signals, we distinguished the linear dichroism signals of ethidium bromide and tryptophan residues in the RecA-DNA-ethidium bromide complex, whereas the conventional (absorption-detected) linear dichroism measurement provides only the sum of the signals because signals overlap each other and that of DNA. We further observed that the tryptophan residue at position 290 of RecA in the RecA-DNA-adenosine-5'-O-(3-thiotriphosphate) complex was oriented parallel to the long axis of the filament, in good agreement with the previous site-specific linear dichroism analysis, and that this orientation was not significantly modified by the pairing of the complementary DNA strand. These results suggest that the pairing reaction occurs without a large structural change of the RecA filament.

Flow injection analysis was used to study the reactions occurring between DNA and certain compounds that bind to its double helix, deforming this and even breaking it, such that some of them (e.g., cisplatin) are endowed with antitumoral activity. Use of this technique in the merging zones and stopped-flow modes afforded data on the binding parameters and the kinetic characteristics of the process. The first compound studied was ethidium bromide (EtdBr), used as a fluorescent marker because its fluorescence is enhanced when it binds to DNA. The DNA-EtdBr binding parameters, the apparent intrinsic binding constant (0.31+/-0.02 microM(-1)), and the maximum number of binding sites per nucleotide (0.327+/-0.009) were determined. The modification introduced in these parameters by the presence of proflavine (Prf), a classic competitive inhibitor of the binding of EtdBr to the DNA double helix, was also studied, determining the value of the intrinsic binding constant of Prf (K(Prf) = 0.119+/-9x10(-3) microM(-1)). Finally, we determined the binding parameters between DNA and EtdBr in the presence of the antitumor agent cisplatin, a noncompetitive inhibitor of such binding. This provided information about the binding mechanism as well as the duration and activity of the binding of the compound in its pharmacological use.

A flow fluorometric approach to study cationic lipoid–DNA complexes is presented. The approach uses standard flow cytometry equipment and common fluorescent dyes (BODIPY and ethidium homodimer-2) to detect both lipoid and DNA content in individual particles. In addition, a procedure that allows determination of whether or not liposomes remain intact is described. The procedure is based on monitoring the retention of a polar tracer that has been preloaded into its aqueous compartment. Sample preparation, instrument setup, data analysis, and methodological limitations are described. Applications of the procedure to cationic lipoid–DNA complexes are described, and illustrations are given for the determination of how the lipoid content, composition, and structure of individual lipoplexes in a population evolve over time, starting at about 1 min after DNA and vesicles are mixed. Analogous procedures can be applied to other heterogeneous particles and supramolecular structures.

We have developed a high-throughput fluorescence anisotropy screen, using a 384-well format, to identify small molecules that disrupt the DNA binding of B-ZIP proteins. Binding of a B-ZIP dimer to fluorescently labeled DNA can be monitored by fluorescence anisotropy. We screened the National Cancer Institute diversity set of 1990 compounds to identify small molecules that disrupt the B-ZIP|DNA complex of CREB, C/EBPbeta, VBP, and AP-1 (FOS|JUND) bound to their cognate DNA sequence. We identified 21 compounds that inhibited the DNA binding of at least one B-ZIP protein, and 12 representative compounds were grouped depending on whether they displaced ethidium bromide from DNA. Of the 6 compounds that did not displace ethidium bromide, 2 also inhibited B-ZIP binding to DNA in a secondary electrophoretic mobility shift assay screen with some specificity. Thermal stability monitored by circular dichroism spectroscopy demonstrated that both compounds bound the basic region of the B-ZIP motif. NSC13778 preferentially binds C/EBPalpha 1000-fold better than it binds C/EBPbeta. Chimeric proteins combining C/EBPalpha and C/EBPbeta mapped the binding of NSC13778 to three amino acids immediately N terminal of the leucine zipper of C/EBPalpha. These experiments suggest that the DNA binding of B-ZIP transcription factors is a potential target for clinical intervention.

A liquid membrane electrode that allows the concentration of ethidium ion (Ed+) to be measured selectively and accurately in the range of 0.1 μM to 5 mM is made. For Ed+ concentrations less than 1 μM or more than 0.1 mM, the trend is no longer linear, and the causes of this behavior are discussed. The mean activity coefficient of ethidium bromide exhibits deviations from the Debye–Huckel limiting law that are interpreted in terms of aggregate formation. The stability constants for Ed 2 2 + and Ed2Br+ are 230 kg mol−1 and 3.0 × 104 kg2 mol−2, respectively. In NaCl solutions, clusters involving up to 4 Ed+ units are detected and their stability constants are evaluated. The intercalation of ethidium into poly(A) · poly(U) in 1 M NaCl is investigated by the above electrode, and the results are compared with those obtained by spectrophotometry. The data are analyzed in terms of Scatchard plots. The potentiometric method is more accurate than the spectrophotometric one at low values of the binding degree (r) where negative deviations from linearity are observed. The deviations are ascribed to a cooperative behavior rather than to artifacts caused by minor systematic errors.

The DNA maintenance enzyme, topoisomerase I, is thought to play crucial roles in all living cells and for this reason inhibitors of this enzyme have been much studied. In this paper we describe a gel electrophoresis method capable of characterizing and quantifying inhibition of topoisomerase I by selected compounds. Inhibitors of topoisomerase I are often associated with intercalative binding to DNA and the method can simultaneously determine intercalative binding (as DNA unwinding) except in the cases where inhibition is prohibitively strong. The method uses closed circular (plasmid) DNA and can separate single-strand nicked, linearized (double-strand nicked), fully relaxed, partially relaxed (topoisomers), and supercoiled forms of the plasmid so that topoisomerase-dependent DNA cleavage (poisoning) can also be determined. By quantifying poisoning, inhibition, and intercalation simultaneously and separately in relation to reference compounds it is possible to make quantitative determinations of these phenomena for comparative purposes. Data for the topoisomerase I inhibitor, luteolin, are presented.

We frequently use competitive PCR in the plateau phase in quantifying DNA species with a small number of cells. However, the basic issues of this method are poorly understood. Here, first we analyze this method theoretically under a generalized condition that competitor and target DNA products accumulate with different amplification efficiencies. We show a theoretical reason that competitive PCR might quantify DNA more accurately during the plateau phase than during the exponential phase. Second, we demonstrate that the theoretical predictions are supported by the experimental results of beta-globin gene amplification using the lysates of human diploid fibroblast WS1 cells. We also demonstrate that we can correctly quantify target DNA by keeping the starting concentration of target DNA close to a constant preset value while using a constant number of PCR cycles and by using WS1 cells as control. Finally, we show the experimental errors in routine measurements of c-myc copy number/cell in human leukemia HL-60 cells with various levels of c-myc multiplication. The number of c-myc copies/cell was determined with an error rate of less than 10%, where agarose gel bands were stained with ethidium bromide for the product quantitation.

The long known toxicity of free chromatin mediated by histones regained attention after discovery of neutrophil extracellular traps (NETs). Free histones from necrotic cells or NETs can damage prokaryotic and eukaryotic cells and are responsible for the aggravation of a growing list of diseases. DNases degrade the toxic chromatin polymer to nucleosomes and efficiently reduce local high histone concentrations. Therefore, DNase activity as a biomarker is of growing interest in basic and clinical research. Here a detailed one-step protocol is presented that allows rapid and sensitive detection of DNases down to 400 fg/μl per reaction based on the detection of fluorescent ethidium bromide/DNA complexes in a 96-well plate reader. The flexible protocol uses an internal standard for background correction and allows convenient and reliable data analysis using common laboratory equipment and chemicals without elaborate preparations. The DNase activity of a sample is clearly defined by substrate amount, incubation time, and (if appropriate) a DNase standard for absolute quantification in Kunitz units per milligram sample protein. Quantitative kinetic determination is possible within less than 1h down to 5 pg DNases/μl per reaction.

A general procedure for the study of conformational transitions of polynucleotides is described. The equilibria between different conformations induced by salt, ethidium bromide, and temperature of poly(dG-dC) · poly(dG-dC) and induced by salt and temperature of poly(A) · poly(U) are investigated using molecular absorption, circular dichroism, and fluorescence spectroscopies. Spectral data obtained from experiments are analyzed by means of a factor analysis method, namely, multivariate curve resolution, which allows possible intermediate states to be detected and the pure spectra and the concentration profiles of all species present in the system to be estimated. This work shows the application of this procedure for the analysis of data matrices obtained in individual experiments but also for the analysis of several data matrices simultaneously.

An assay for quantitation of plasmid copy numbers in bacterial cell cultures has been developed and validated. The method combines isolation of total bacterial DNA (including both plasmid and genomic DNA), running a series of twofold dilutions of total DNA in an agarose gel followed by ethidium bromide staining, and subsequent scanning of the gel picture negatives. We have developed a novel set of rules for integration of the scan data that allows us to achieve high assay precision, accuracy, and sensitivity. The assay validation results were as follows: intra- and interassay precision with %CV of 8.2-9.9 and 7.1-9.8%, respectively; ruggedness with %CV of 9.3-17.5%; spike recovery of 80-102%; and sensitivity of 1 plasmid copy per genome.

The highest sensitivity nucleic acid gel stains developed to date are optimally excited using short-wavelength ultraviolet or visible light. This is a disadvantage for laboratories equipped only with 306- or 312-nm UV transilluminators. We have developed a new unsymmetrical cyanine dye that overcomes this problem. This new dye, SYBR Gold nucleic acid gel stain, has two fluorescence excitation maxima when bound to DNA, one centered at approximately 300 nm and one at approximately 495 nm. We found that when used with 300-nm transillumination and Polaroid black-and-white photography, SYBR Gold stain is more sensitive than ethidium bromide, SYBR Green I stain, and SYBR Green II stain for detecting double-stranded DNA, single-stranded DNA, and RNA. SYBR Gold stain's superior sensitivity is due to the high fluorescence quantum yield of the dye-nucleic acid complexes ( approximately 0.7), the dye's large fluorescence enhancement upon binding to nucleic acids ( approximately 1000-fold), and its capacity to more fully penetrate gels than do the SYBR Green gel stains. We found that SYBR Gold stain is as sensitive as silver staining for detecting DNA-with a single-step staining procedure. Finally, we found that staining nucleic acids with SYBR Gold stain does not interfere with subsequent molecular biology protocols.

Photography or electronic image acquisition is required to document results obtained from staining protein gels with the fluorescent SYPRO dyes. We found that, when using Polaroid type 667 or 57 instant films, the choice of optical filter combination and photographic exposure time strongly influences protein detection sensitivity limits. Ultraviolet light-blocking Kodak Wratten No. 2A and 2B gelatin filters autofluorescence when illuminated at 300 nm. The use of these filters in combination with Wratten No. 22 or 25 filters or SYPRO gel photographic filters gives rise to increased background signals, which for long photographic exposures can obscure signals due to protein bands. Surprisingly, the use of these same ultraviolet lightblocking filters enhanced the protein detection sensitivity obtained with short photographic exposures. Under the conditions tested, we found minimal differences in performance for Polaroid type 667 and 57 films.

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.

The ribonuclease (RNase) activity of ribonucleases has been assayed by observing the change in fluorescence intensity of ethidium bromide on binding with yeast RNA. The binding of EtBr with RNA was monitored via UV-vis and fluorimetric methods. The degradation of RNA by RNase A was monitored by the change in fluorescence emission intensity of ethidium bromide at 600nm on excitation at 510nm. The ribonucleolytic activity of RNase A and angiogenin at various pH values was determined by this method. From this technique we have also determined the macroscopic pKa values of active site residues of these enzymes. This assay permits the evaluation of the catalytic efficiency of enzymatic proteins ranging from high ribonucleolytic activity to low ribonucleolytic activity toward the natural substrate RNA.

A method for detecting the activity of ribonuclease inhibitors (RIs) after nondenaturing polyacrylamide gel electrophoresis and isoelectric focusing was developed. Both types of electrophoresis were performed using vertical slab polyacrylamide gels in the presence of dithiothreitol and in a sealed system. In each system, purified 50 kDa human RI was visualized as a single band by immunoblotting with a specific antibody. RI activity in the polyacrylamide gel slab was detected by sandwiching the gel slab between a cellulose acetate membrane moistened with a solution of bovine pancreatic ribonuclease A and a dried agarose film sheet containing substrate yeast RNA plus ethidium bromide and incubating at 37°C. The ribonuclease penetrated the polyacrylamide gel and digested the substrate RNA in the agarose film. However, if an RI was present in the gel, the enzyme was inactivated by complex formation. Fluorescent bands corresponding to RIs were observed on a dark background under ultraviolet light. This activity staining had a high sensitivity allowing detection of less than 0.6 units of mammalian RIs (corresponding to 5 ng of purified human RI) and produced a sharp band which compared favorably with that obtained on immunoblotting. These electrophoretic techniques appear useful for the investigation of RIs in heterogeneous biological samples.

Mobilities of DNA, fluorescently labeled with ethidium homodimer (EtD), and normalized to the mobility of the 50-bp fragment [Rf(50)], are constant with time of electrophoresis, permitting one to conduct studies on DNA mobility in an automated electrophoresis apparatus with fluorescence detection. DNA mobility decreases with an increasing binding density of ethidium homodimer, as previously reported by others and expected since the dye decreases both the net charge and the conformational flexibility of the DNA. However, it was found that this effect of ethidium binding on electrophoretic mobility becomes undetectable at binding densities less than 1 EtD/40 bp. This implies that for the purposes of electrophoretic analysis in an apparatus with fluorescence detector, DNA with ethidium homodimer intercalated at dye-DNA ratios less than 1/40 bp behaves like unlabeled DNA and may be assumed to be in a conformation similar to that of the unliganded DNA. Necessarily, the low labeling ratio lowers the sensitivity of detection and, in particular, increases the load requirement for a full-scale band height required for the analysis of bandwidth and band shape during electrophoresis.

A buffer containing 7 M urea was used to successfully prevent DNA degradation of in situ damaged fish skin and resulted in reproducible high molecular weight DNA. This treatment in combination with a quantitative gel electrophoresis technique permitted estimation of small changes induced in unlabeled DNA per unit molecular weight after treatment with a DNA damaging agent (e.g., ultraviolet irradiation).

An automated two-dye flow injection analysis system to quantitate DNA and RNA in crude extracts of tissues is described. The method uses the fluorochrome dyes ethidium bromide and Hoechst 33258. DNA concentration is determined directly from its fluorescence in Hoechst dye. RNA is estimated from fluorescence in ethidium bromide after subtraction of the fluorescence due to DNA. This method has several advantages: a simple extraction procedure, a low detection limit (0.01 micrograms DNA and 0.10 micrograms RNA), automation, and a high sample throughput.

The electrochemically controlled release of the ethidium cation from the surface of carbon paste composite electrode was demonstrated using laser-induced fluorescence detection. The electrode contained an ethidium tetracyanoquinodimethane salt. The following experimental parameters were varied in order to optimize the fluorescence intensity: carbon paste composition, electrode potential, voltage pulse time, and laser power. In the presence of calf thymus DNA the fluorescence signal from the diffusion layer was linearly dependent on the logarithm of the DNA concentration over the range from 0.1 to 104 ppb.

Accurate quantitation of the distribution of DNA digested to completion by a restriction endonuclease and separated in an electrophoretic gel is greatly facilitated by electronic imaging systems employing charge-coupled device detectors. Such quantitation permits the extraction of more accurate values of the lengths of both the parent molecular species and the fragments produced by a restriction endonuclease. We describe a method that determines the number of components in bands containing two or more daughter fragments that are of similar length and hence not resolved by gel electrophoresis. We also report a constrained deconvolution procedure based on the equal number of daughter molecules in a terminal digestion by a restriction endonuclease that resolves the distribution of each of the overlapping species in a composite band on a gel and hence increases the accuracy of the lengths assigned to each.

A modification, using S1-nuclease, of a simple and sensitive fluorometric assay with ethidium bromide was developed for the measurement of cellular DNA interstrand crosslinking induced by bifunctional alkylators. Cells are lysed and treated with proteinase K and sodium dodecyl sulfate followed by extensive dialysis to yield intact high-molecular-weight DNA, free of contaminating proteins, on which the crosslink assay is then performed. The assay depends on the differential binding of ethidium bromide to single- and double-stranded DNA. Because of the higher ethidium bromide binding capacity of double-stranded DNA, the fluorescence retained after a heating/cooling cycle is directly proportional to the drug-induced cellular DNA interstrand crosslinking. We demonstrate that the sensitivity of this assay can be increased up to fourfold by including an S1-nuclease digestion step. This modified technique is simple and suited to the quantitation of low levels of DNA-interstrand crosslinking in cells.

DNA self-assembly with polycations produces nanoparticles suitable for gene delivery, although there is no standard methodology to measure particle formation and stability. Here we have compared three commonly used assays, namely, light scattering, inhibition of ethidium bromide fluorescence, and modified electrophoretic mobility of DNA. Analysis by light scattering and loss of ethidium bromide fluorescence both showed poly(l-lysine) (pLL)/DNA nanoparticles form over the lysine/phosphate ratio range 0.6–1.0, although retardation of DNA electrophoretic mobility commenced at lower lysine/phosphate ratios. This probably indicates that the first two assays monitor DNA collapse into particles, while the electrophoresis assay measures neutralization of the charge on DNA. Gel analysis of the complexes showed disproportionation during nanoparticle formation, probably reflecting cooperative binding of the polycation. The assays were used to examine stability of complexes to dilution in water and physiological salts. Whereas all pLL/DNA nanoparticles were stable to dilution in water, the presence of physiological salts provoked selective disruption of complexes based on low-molecular-weight pLL. Polyelectrolyte complexes for targeted application in vivo should therefore be based on high-molecular-weight polycations, or should be stabilized to prevent their dissociation under physiological salt conditions.

We describe an approach to creating a new class of luminophores which display both long wavelength emissions exceeding 600 nm and long lifetimes. These luminophores are based on resonance energy transfer (RET) from a long lifetime donor to a short lifetime but long wavelength acceptor. We demonstrated the possibility of obtaining these desirable spectral properties using donors and acceptors noncovalently bound to DNA. The donor was a ruthenium (Ru) metal–ligand complex in which one of the diimine ligands intercalated into double-helix DNA. The acceptors were either nile blue, TOTO-3, or TO-PRO-3. Upon binding of the acceptor to donor-labeled DNA, we found that the acceptor quantum yield was remarkably enhanced so that the wavelength-integrated intensities of the donor and acceptor bound to DNA were many-fold greater than the intensity of the donor and acceptor alone when separately bound to DNA. The origin of this effect is efficient energy transfer from the donor. Under these conditions the effective overall quantum yield approaches that of the acceptor. Importantly, the increased quantum yield can be obtained while maintaining usefully long apparent acceptor lifetimes of 30 to 80 ns. The effect of an increased quantum yield from a low quantum yield donor may find use in assays to detect macromolecular binding interactions. These results suggest the synthesis of covalently linked donor–acceptor pairs with the desirable spectral properties of long wavelength emission, high quantum yield, and moderately long lifetimes for gated detection.

Two-dimensional electrophoresis in agarose gels separates DNA-restriction fragments not only by molecular weight but also according to their AT-cluster content. The method produced genus-specific spot patterns of multicopy DNA fragments of grains as well as spot patterns of highly repetitive DNA fragments of ciliates, demonstrated for barley, spelt, and Tetrahymena. Further investigations in regard to their specificity by hybridization with three other grain species (wheat, oat, and rye) and three ciliate species (Tetrahymena thermophila, Tetrahymena pigmentosa, and Tetrahymena borealis) were performed. The DNA samples from spelt and Tetrahymena were demonstrated to be genus specific for Triticum and species specific for Tetrahymena pyriformis, respectively.

The commercially available copolymer of 10 mol% acrylic acid and polyethylene is easily formed into a nonfluorescing, non-polynucleotide-adsorbing film. The film has surface carboxylate functions whose concentration can be increased by heating to 80 degrees C in 30% NaOH. The carboxylate groups will react at pH approximately 7 with commercially available, oligo-DNA, 2-8 ng/microl, that has been synthesized with a C(12)-alkylamino tail on the 5'-end. The reaction is mediated with water-soluble carbodiimide reagent and is assumed to result in a primary amide bond between the polymer film and the modified oligo-DNA. The tethered oligo-DNA retains its hybridization activity, and its surface concentration is sufficient to permit qualitative, labelless detection of hybridized target by fluorescence after brief staining with ethidium bromide. The film is used to detect Shiga-like toxin gene II (SLT-II) from Escherichia coli O157:H7 after asymmetric, capillary, PCR amplification, and a 4-h hybridization. Captured target may be removed from the film using distilled water, after which the film can be used again without noticeable loss of activity. The method provides relatively rapid detection of PCR amplimers without having to use molecular labels, or surface-fouling agents.

In this paper, we report a fast, simple, and reproducible staining protocol for nucleic acids in agarose gels with a sensitivity in the order of 10 pg/mm2. It took only three steps: fixation, incubation with silver ions, and development of the gels (total time 50 min). The resulting calibration curves (area vs ng of loaded DNA) after a densitometric scanning of agarose gels stained with this procedure were linear up to 50 ng of double-stranded DNA. We found this method suitable for routine laboratory use and especially appropriate for densitometric analysis due to homogeneous background development. Furthermore, it avoids the pretreatment and/or drying steps proposed by other authors for agarose gels.

A new series of high-affinity cyanine dyes was tested for the visualization of the dynamics of single DNA molecules through a fluorescence microscope. In particular, YOYO-1 (1,1'-(4,4,7,7-tetramethyl-4,7-diazaundecamethylene)- bis-4-[3-methyl-2,3-dihydro-(benzo-1,3-oxazole)- 2-methylidene]-quinolinium tetraiodide) forms a very stable, highly fluorescent complex with double-stranded DNA and dramatically improves the quality of the images. We have characterized the factors affecting the signal/background in the imaging of single DNA molecules by fluorescence microscopy and compared the results obtained using YOYO-1 with those obtained using standard fluorescent dyes like ethidium bromide or acridine orange.

A method to detect leakage of water-soluble intercalators such as ethidium bromide and propidium iodide from lipomes (egg-lecithin and egg-lecithin/cholesterol) by means of fluorescence is presented. The addition of excess DNA outside the liposomes brings about intercalation of released dye with the DNA. The increase in fluorescence intensity that results from the dye–DNA complex is used to detect the degree of dye release. Titration and surfactant-induced leakage measurements display the suitability of the method. Spontaneous leakage measurements show that propidium has much lower leaking rates than those of ethidium. This is probably due to the less lipophilicity of propidium as estimated from the partition coefficients between octanol and medium. Stabilizing the egg-lecithin membranes with cholesterol strongly reduces the leaking rates for ethidium while no effect is detected for the slow leaking rates of propidium. Lowering the temperature from 25 to 6°C results in lower leaking rates for both dyes.

A microtiter plate assay was developed to quantitate the nuclease activity of the extracellular Serratia marcescens endonuclease under different buffer conditions. Substrate cleavage was followed as decrease in ethidium/DNA fluorescence using a uv-transilluminator and a video documentation system. Time courses of DNA cleavage were recorded and cleavage rates determined very precisely within a factor of 1.2. The assay has a linear dynamic range covering three orders of magnitude of nuclease activity and can be carried out very quickly within a few minutes. It can also be used with RNA as substrate. With appropriate modifications it should be possible to adapt this assay for other enzymatic reactions which are accompanied by changes in absorbance or fluorescence.

Visualization of DNA in electrophoretic gels typically requires UV radiation and the fluorescent dye ethidium bromide. Alternatively, we report here that by inclusion of visible dyes in standard agarose gels, DNA bands are observable in ambient light as they are separating. Such bands can be directly recovered from gels (approximately 50% yield) and used in standard enzymatic reactions (ligation, endonucleolytic cleavage, random labeling, PCR, and cycle-sequencing) without purification. Of 14 common commercially available stains that could visualize fractionating DNA, Nile blue was chosen for more extensive analysis as it gave the sharpest and most persistent bands and is not known to be toxic. Bands containing greater than 40 ng DNA could be detected by direct visual inspection of gels during electrophoresis. Drying the gels increased sensitivity to 4 ng. We describe relevant molecular features of these dyes and detail simple assays that may be employed to find other useful, and perhaps superior, dyes. This method also lends itself to situations in which easy visualization and convenience of DNA electrophoresis are important, such as classroom demonstrations.

We have developed a method for the comparison of quantitative differences between nucleic acid samples run on agarose gels. It is useful for the analysis of digested genomic DNA and RNA preparations, as well as for clamped homogeneous electric field gel analysis of damaged chromosomal DNA. The technique utilizes a standard color charge-coupled device video camera, a microcomputer, and commercially available software. While not as elaborate as other image analysis systems, our method is suitable for many purposes and can be set up for a relatively low cost. In principle, any system capable of recording 24-bit color scans and measuring pixel color intensity could be used.