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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.

Differential display, suppression subtractive hybridization and other techniques for identification of differentially expressed genes produce fragments of cDNA from mRNAs whose differences in abundance must be verified. This report describes a relative multiplex RT-PCR assay that facilitates the analysis of large numbers of samples for differences in mRNA abundance without the use of radioactivity or blotting. The species of interest is co-amplified with 18S rRNA over a range of cycles followed by electrophoresis through ethidium bromide-agarose gels. Intensities of the bands of interest, normalized for 18S band intensities, are plotted as a function of cycle number. Regression equations fitted to the curves are used to calculate the number of cycles necessary for each sample’s normalized signal to reach a threshold intensity. Differences between samples in the number of cycles required to reach that threshold reflect differences in the original abundances of those species. A comparison with results previously obtained using northern blots showed that relative differences as small as 20% and as large as an order of magnitude are accurately detected. The simplicity of the assay allows its routine application in both research and teaching laboratories.

RNA gel analysis is essential for quality assessment of RNA preparations for subsequent analysis such as microarrays and real-time PCRs. The routinely used standard electrophoresis of RNA through formaldehyde-containing agarose gels is not only labor-intensive and time-consuming, but also involves sizeable quantities of hazardous materials. Above all, it is not sensitive, requiring more than 1 µg of RNA for the assay. Current gene expression profiling with microarrays and real-time PCR often involves limiting amounts of RNA. It is therefore important to have a more sensitive way to analyze RNA. Here we report an improved ethidium bromide-based RNA gel analysis system with our Superload buffer that increases sensitivity to 12.5 ng of total RNA and allows RNA analysis on a regular native Tris-acetate EDTA (TAE) agarose gel.

GelStar nucleic acid gel stain can be used for sensitive fluorescent detection of both double-stranded (ds) and single-stranded (ss) DNAs, oligonucleotides and RNA in gels. The stain can be added to agarose gels at casting for immediate imaging after electrophoresis or can be used after electrophoresis with both agarose and acrylamide gels. GelStar stain is highly fluorescent only when bound to nucleic acids thus giving superior signal-to-noise ratios and obviating the need to destain the gel. The detection limits of GelStar strain are 20 pg for dsDNA, 25 pg for ssDNA and 10 ng for native or glyoxal-treated RNA.

Methods for detecting and measuring the success of nucleic acid sequence amplifications can be developed by detecting the by-products of amplification procedures. One method includes the detection of inorganic phosphate (Pi) during or on completion of the PCR. The method requires modification of assay conditions to prevent thermal- and template-independent enzymatic activity from nonspecifically hydrolyzing dNTPs. Detection of Pi by the traditional Fiske-SubbaRow method provides a sensitivity similar to ethidium bromide staining of amplified products. The method offers a simple and rapid assay for amplified nucleic acids and can be useful in assays where confirmation of the amplified DNA product is not essential.

A rapid and easy procedure for preparing Northern blots is described. RNA is denatured with glyoxal in the presence of ethidium bromide and glycerol, then electrophoresed through agarose in a buffer that does not require recirculation. Without any additional washes, the RNA is vacuumblotted to a nylon membrane in NaOH, which simultaneously removes the glyoxal adducts. All of these steps plus prehybridization of the filter and addition of a digoxygenin-labeled probe can be completed in one day. Using standard procedures to wash the filters and detect the probe, the entire procedure can be completed within two days.

A method is presented for the analysis of fluorescently labeled random amplified polymorphic DNA (FRAPD) fragments. A DNA sequencer and collection and analysis software were used to estimate the sizes of DNA fragments based on their mobilities relative to in-lane size markers. This allowed confident identification and comparison of FRAPD markers both within and between polyacrylamide gels. In comparison with analysis of RAPD products using ethidium bromide-stained agarose gels, fluorescent analysis improved the sensitivity, resolution and precision of sizing of RAPD products of about 50–2100 bp. FRAPD fragments produced from amplification of zebrafish DNA are informative as genetic markers that segregate with Mendelian inheritance. FRAPD analysis was found to be very efficient for identifying new DNA polymorphisms.

To improve Salmonella detection, we developed nonradioactive hybridization assays of amplified products from pure Salmonella cultures. Biotin-labeled PCR products were trapped by internal probes covalently bound to CovaLink-NH MicroWells and detected by colorimetric or chemiluminescent enzymatic reactions. The sensitivities of colorimetric assays using peroxidase and alkaline phosphatase were similar to those obtained with an ethidium bromide-stained agarose gel; both procedures allow the detection of 50 Salmonella cells. Chemiluminescence was 10-fold more sensitive than colorimetry.

Genotyping (typing of genetic variation) typically involves PCR followed by an allele-specific oligonucleotide-binding assay, restriction enzyme digest or direct check of the outcome of a PCR designed to distinguish genotype. Electrophoresis can resolve "bound" from "free" oligonucleotide, as well as resolve PCR fragments and digests, but it is traditionally regarded as cumbersome and laborious in comparison with solution assays. Here we describe simple horizontal polyacrylamide gels which can receive a 96-well array of samples directly, which can be stacked in tanks and which are bound to a robust support of glass. The line of electrophoresis is on a 71.6 degree diagonal relative to the columns of the array (microplate array diagonal gel electrophoresis [MADGE]). Several thousand reactions can conveniently be analyzed in a shoebox-sized apparatus in a couple of hours. High resolution is achieved in the range of 20-1000 bp, information processing is simplified and automation is possible.

We have developed a rapid and sensitive method to analyze double-stranded DNA by capillary electrophoresis equipped with a laser-induced fluorescence detector. Using ethidium bromide as an intercalator in polyacrylamide gel-filled capillaries along with a green He/Ne laser source (excitation: 543 nm; emission: 600 nm), we found the detection sensitivity for a single DNA-ethidium bromide complex was in the picogram level with a resolution of one base pair separation between two DNA fragments. In the case of the separation of a phi X174 DNA-HaeIII digest, sizes from 72 to 1353 bp were well resolved within 12 min. For the analysis of PCR-amplified DNA, low levels of amplified DNA fragments could be detected that could not be visualized in agarose gel after standard ethidium bromide staining. Considering the high resolving power and sensitivity, we believe that this method can be a useful tool for the analysis of double-stranded DNA especially for PCR-amplified DNA fragments.

A PCR assay has been optimized for the detection of one or a few molecules of a translocation-containing human DNA sequence in the presence of a vast excess (7 micrograms) of the normal human genome. This procedure avoids blot hybridization by the use of two rounds of PCR with 20-22 cycles of amplification per round and by the replacement of one of the two primers from the first round of PCR with a different primer in the second round (semi-nested PCR). We demonstrate that very low numbers of the target DNA molecules can be quantitated by this semi-nested PCR. This method can be used to detect a single DNA molecule from one mutant cell displaying a translocation between the bcl-2 proto-oncogene region and a JH immunoglobulin gene sequence [t(14;18)] in a background of normal human DNA from 10(6) cells.

Agarose gel electrophoretic separation of a lambda/HindIII DNA marker containing detectable fragments of 23 to 2 kb was carried out in the conventional submarine apparatus and in the horizontal gel slab apparatus of Wieme, using identical samples, agarose, gel width and procedure for ethidium bromide staining. In the Wieme apparatus, the gel on its microscope slide support is immersed in an immiscible solvent such as petroleum ether or silicone oil. Although band resolution and speed of migration are equivalent between gels run in the two systems, the relative fluorescence intensity of the ethidium bromide-stained bands is substantially more responsive to an increase in DNA length in the Wieme gels than in the submarine gels. The predicted relative fluorescence is by an average factor of 0.5 less than that observed after electrophoresis in the Wieme apparatus but is by an average factor of 3.4 more than that observed on bands derived from the submarine technique.

A spectrofluorometric assay to detect hybridization of oligodeoxynucleotides is described. Ethidium dimer, which yields significant fluorescence enhancement when intercalated between adjacent base pairs, was employed as the probe. Maximum enhancement of fluorescence occurred at equimolar ratios of p(dA)20.p(dT)20, while negligible fluorescence occurred for either of these chains when not in the presence of the other. Base-paired oligonucleotides at concentrations as low as 20 nM nucleotide could be detected, and the assay was linear over the tested range of 20 to 350 nM nucleotide. The method was used to measure the temperature-dependent melting and hybridization of two self-complementary heteropolymers and the hybridization of a self-annealing (dG)20 preparation.

Carry-over contamination from PCR products can yield persistent false-positive results leading to significant problems in the resolution of true positive results. Commercial kits used in many laboratories help prevent carry-over contamination. We present data on the effects of dUTP substitution on the hybridization properties of amplified DNA using alkaline phosphatase conjugated oligonucleotide probes. We observe a pronounced depression in hybridization signal intensity in some dUTP-substituted PCR products. The magnitude of the decreased hybridization signal intensity appears proportional to both the dUTP concentration in the amplification reaction and the fraction of thymidylate residues in the probe binding site. The hybridization signal is nearly eliminated from PCR products synthesized in the presence of dUTP only and where the probe binding site is particularly rich in thymidylate residues. The decrease in hybridization signal is not always restored with less stringent hybridization conditions. Conditions that permit efficient hybridization and detection of bound probe but do not compromise carry-over protection are discussed.

Current methods of localization of chromosomal antigens on polytene chromosomes of Drosophila melanogaster salivary glands by indirect immunofluorescence require comparison of two microscopic images of the same nucleus--a phase contrast or bright field image to visualize the chromosomes and a fluorescent image to locate the antibody. We have found that inclusion of the DNA-intercalating agent ethidium bromide in the mounting medium makes banded polytene chromosomes visible under epifluorescent illumination, eliminating the need for two images. The banding of the polytene chromosomes is clear enough to locate specific bands.

Supercoiled and relaxed DNA were resolved electrophoretically in the presence of 0.5 micrograms/ml ethidium bromide. Under these conditions the Gaussian distributions of topological isomers of both supercoiled and relaxed DNA migrated as discrete bands. The separation of these DNAs was optimized by varying the concentration of electrode buffer. Electrophoresis in the presence of 160 mM Tris-acetate, pH 8.3, 4 mM EDTA resulted in a 20-fold increase in the separation of relaxed and supercoiled DNA relative to electrophoresis in 40 mM Tris-acetate, pH 8.3, 1 mM EDTA.

During the development of a micro-method for the quantitative analysis of gene expression, we observed that the sensitivity for detection of PCR-amplified product was higher when silver staining was used, compared with either ethidium bromide staining (as reported previously) or Southern hybridization with 32P-labeled oligonucleotide probe. This observation may have an important impact on the analysis of RNA-PCR-amplified signal for the quantitation of mRNA expression, simply by densitometric scanning of silver-stained polyacrylamide gel. We believe that this is the first report to show such a comparison, demonstrating the greater sensitivity of silver staining compared with either ethidium bromide staining or Southern hybridization utilizing an oligonucleotide probe prepared by 3'-end labeling with 32P-dATP.

In order to study quantitative gene expression with Northern blots, it is important to have an internal standard that can be used to verify even loading or to correct for uneven loading between lanes. In this study it is shown that two-dimensional quantitation of ethidium bromide-intercalated 28S rRNA fluorescence can be used for such standardization. It was found that the film response of the fluorescence was linear with respect to total loaded RNA in the range of 2.5-12.5 micrograms RNA under the conditions used, after which the linear relationship falls off. This method eliminates the use of radiation for internal standardization of Northern blots.

A high-sensitivity, laser-excited confocal fluorescence gel scanner has been developed and applied to the detection of fluorescently labeled DNA. An argon ion laser (1-10 mW at 488 nm) is focused in the gel with a high-numerical aperture microscope objective. The laser-excited fluorescence is gathered by the objective and focused on a confocal spatial filter, followed by a spectral filter and photodetector. The gel is placed on a computer-controlled scan stage, and the scanned image of the gel fluorescence is stored and analyzed in a computer. This scanner has been used to detect DNA separated on sequencing gels, agarose mapping gels and pulsed field gels. Sanger sequencing gels were run on M13mp18 DNA using a fluoresceinated primer. The 400-microns-thick gels, loaded with 30 fmol of DNA fragments in 3-mm lanes, were scanned at 78-microns resolution. The high resolution of our scanner coupled with image processing allows us to read up to approximately 300 bases in four adjacent sequencing lanes. The minimum band size that could be detected and read was approximately 200 microns. This instrument has a limiting detection sensitivity of approximately 10 amol of fluorescein-labeled DNA in a 1 x 3-mm band. In applications to agarose mapping gels, we have exploited the fact that DNA can be prestained with ethidium homodimer, followed by electrophoresis and fluorescence detection to achieve picogram sensitivity. We have also developed methods using both ethidium homodimer and thiazole orange staining which permit two-color detection of DNA in one lane.(ABSTRACT TRUNCATED AT 250 WORDS)