An Eye for a Dye

Nucleic Acid Dyes and Stains DNA molecular weight markers stained with Molecular Probes' SYBR Green I Researchers working with DNA often have two fundamental questions: "How much DNA have I got?" and "What size is it?" Large quantities of DNA can be visualized even without staining or special illumination: The schlieren lines--refractive index changes at the boundaries of concentration differences--reveal the positions of the bands. However, this method is impractical for most applications. Fortunately there are numerous chemicals that show an increased fluorescence upon binding to nucleic acids, allowing easy detection using a light source and the appropriate filters. Ethidium bromide (2,7-diamino-10-ethyl-9-phenylphenanthridinium bromide; EtBr) has traditionally been used for staining DNA and RNA in gels (and here) following procedures first published in the early 1970s.1 While the procedures for using EtBr are simple, EtBr is fairly toxic. The material safety data sheet (MSDS) on EtBr does not explicitly list it as a carcinogen, but the stain does have mutagenic activity, and anything that binds DNA should be handled with care (msds.pdc.cornell.edu). Although some cavalier researchers claim that dilute EtBr solutions are harmless ("The EtBr is bound up by the DNA in the dead skin cells and never penetrates deep enough to do any damage!"), wearing gloves when working with EtBr is usually considered essential. The MSDS claims EtBr "may enter the body through inhalation, ingestion, and eye and skin contact." Spills must be carefully cleaned up, and weighing solid material--the cheapest way to obtain concentrated stock solutions--can be a static-filled, harrowing experience requiring a good mask and fume hood. After use, EtBr solutions require special decontamination methods. Concentrated solutions (such as 10 mg/ml stocks) require dilution to <0.5 mg/ml and prolonged incubation with hypophosphorous acid (itself very corrosive and hazardous) and sodium nitrate, which reduces the mutagenicity by ~200-fold.2 Dilute solutions (such as gel buffers containing ~0.5 µg/ml EtBr) should be decontaminated using amberlite resin or activated charcoal, as the traditional methods of neutralization with bleach result in the formation of other mutagenic compounds.3,4 Although its excitation maximum is around 500 nm, EtBr also absorbs ultraviolet light at 254, 302, and 366 nm. Fluorescent emission is at 590 nm, and because EtBr bound to DNA has a greater fluorescence quantum yield than free dye, EtBr:DNA complexes are readily visualized. At saturation, one molecule of EtBr will intercalate into approximately every second base pair in double stranded DNA (dsDNA), making EtBr staining surprisingly sensitive. Although background fluorescence in agarose can be significant, with complete destaining of the gel, sharp bands containing as little as 10 ng of DNA can be discerned by eye. With careful photography, bands containing ~1 ng of dsDNA can often be detected. The efficiency of intercalation into single stranded DNA (ssDNA) and RNA is reduced, but EtBr can also be used to examine these materials. Illumination of EtBr-stained gels is generally done at 302 nm.3 This is a compromise between the reduced quantum yield at 366 nm and the increased nicking at 254 nm. Nicking can be a problem when the DNA bands are to be excised from the gel for downstream applications. A Kodak Wratten® 22A filter eliminates most of the illuminating light while allowing most of the fluorescence to reach the camera. In many labs, the Polaroid cameras traditionally used for this application are being replaced with digital cameras or imaging systems. Over the past few years, many alternatives to EtBr have become commercially available. Many are less hazardous, more sensitive, or both. Ignoring the myriad of covalent labeling, amplification, or hybridization approaches, this article will take a look at some of the newer nucleic acid dyes and stains available. There are two instances when detecting or quantifying DNA in a sequence-independent fashion are important: determining solution concentrations and examining size-resolved nucleic acids on gels. RNA stained with Bio-Rad's Radiant Red. Manufacturers and distributors of nucleic acid stains (other than EtBr) Accurate Chemical and Scientific Corp. (800) 645-6264 www.accuratechemical.com American Bioanalytical Inc. (800) 443-0600 www.americanbio.com Amersham Pharmacia Biotech Inc. (800) 526-3593 www.apbiotech.com Amresco Inc. (800) 829-2805 www.amresco-inc.com Bio-Rad Laboratories (800) 424-6723 www.discover.bio-rad.com BioWhittaker Inc. (800) 341-1574 www.bmaproducts.com bioWORLD (800) 860-9729 www.bio-world.com Carolina Biological Supply Co. (800) 334-5551 www.carolina.com Crescent Chemical Co. (800) 645-3412 www.crescentchemical.com Invitrogen Corp. (includes Novex) (800) 955-6288 www.invitrogen.com ISC BioExpress (800) 999-2901 www.bioexpress.com Merck Ltd. 44 1202 669700 www.merck-ltd.co.uk Midwest Scientific (800) 227-9997 www.midsci.com Molecular Probes Inc. (800) 438-2209 www.probes.com Pierce Chemical Co. (800) 874-3723 www.piercenet.com Promega Corp. (800) 356-9526 www.promega.com Roche Molecular Biochemicals (800) 262-1640 biochem.roche.com Sigma (800) 325-3010 www.sigma-aldrich.com VWR Scientific Products (800) 932-5000 www.vwrsp.com The Lives of a Gel Staining a gel with EtBr is simple. EtBr can be added to the electrophoresis running buffer or to the gel itself before casting. The gel can then be transferred directly to a UV illuminator (transillumination or top lighting both work well) and examined immediately. Many researchers prefer to keep their gel tanks EtBr-free, in which case the gel can be transferred to a solution of 0.5 µg/ml EtBr after electrophoresis and gently shaken for 15-20 minutes. Background fluorescence can be eliminated by a 20-60 minute destain in running buffer or water. Greatly increased sensitivities over EtBr can be achieved using silver stains. These rely on the reduction of ionic to metallic silver and can achieve sensitivities greater than ~1 pg of nucleic acid per band: as good as many radiolabeling techniques and even adequate for DNA sequencing. Efficient silver staining is a lengthy process, however, typically requiring the following steps: fixation, multiple washes, pretreatment, multiple washes, silver staining, development, fixation, and more washes. The technique also employs hazardous materials, such as formaldehyde, and because it requires large volumes of silver nitrate solution, it is usually considered to be too expensive for routine gel work. A group of stains based on unsymmetrical cyanine dyes have been gaining acceptance for DNA and RNA gel staining in recent years. The most popular are the SYBR dyes made by Molecular Probes of Eugene, Ore. SYBR Green I dye, for staining DNA in gels, is probably the most widely used. Ames mutagenicity tests show that SYBR Green I stain is far less mutagenic than EtBr.5 In other tests, EtBr induced frameshift mutations at rates close to 70-fold above controls. Rates for SYBR Green I stain were a little over twice those seen with controls in this strain. In point-mutation indicator strains, both EtBr and SYBR Green I stain resulted in reversion rates that were approximately twice the control level. Another unsymmetrical cyanine dye with sensitivity on par with that of SYBR Green I is Vistra Green™ from Amersham Pharmacia Biotech of Uppsala, Sweden. Vistra Green is made specifically for use with fluorescence scanning systems from Molecular Dynamics of Sunnyvale, Calif. Dyes that bind DNA fall into three mechanistic categories. The intercalating dyes, typified by EtBr and including other commonly used dyes such as propidium iodide, bind DNA by inserting "flat" chemical moieties between adjacent bases. Minor groove binders, such as the Hoescht series and DAPI, have elongated structures that wrap around the DNA, slotting into the minor groove. Although the mechanism by which SYBR Green I binds to DNA is not known, results from Ames tests imply that it is probably not through base intercalation. SYBR-like dyes are also more sensitive than EtBr. The EtBr:DNA complex has a fluorescence quantum yield of ~0.15 (about 15 percent of the excitation light is converted to fluorescence). In contrast, SYBR Green I stain complexed with DNA has a fluorescence quantum yield of ~0.8. Under optimal conditions, SYBR Green I stain allows detection of ~60 pg of dsDNA in a well-defined band. SYBR Green I stain shows higher sensitivity than EtBr using a standard 302 nm transilluminator and even greater sensitivity using 254 nm shortwave illumination. However, as one of the advantages of SYBR Green I stain is compatibility with enzymatic reactions, illumination at 254 nm may not be desirable--it leads to nicking of the DNA. Emission is at 521 nm, close to that of fluorescein. Because excitation at 494 nm is also efficient, SYBR Green I stain is compatible with many types of scanners and other instruments. For conventional photography, several special filters are available from Molecular Probes, or a Kodak Wratten #15 will also work. It should be noted that the best filters for Polaroid film may not be optimal for CCD cameras; camera manufacturers should be able to help optimize most systems. While SYBR Green I stain does have a large quantum yield, this is only part of the sensitivity picture. The fluorescence enhancement upon binding nucleic acid is also important. EtBr has about a 30-fold enhancement upon binding dsDNA; that is, the quantum yield of the EtBr:DNA complex is about 30 times as great as that of unbound EtBr. SYBR Green I stain improves on this by more than an order of magnitude. In addition to detecting smaller quantities of DNA, dyes that have large fluorescence enhancements upon binding lead to significantly lower background signals from unbound dye; increased signal to background ratio contributes to their high sensitivity. Although its quantum yield is less (around 0.6), when combined with a fluorescence enhancement of ~1000-fold, SYBR Gold stain takes the sensitivity prize. Under optimal conditions, this stain will detect about 25 pg of DNA in a sharp band, approaching or even exceeding the sensitivity of silver staining or radiolabeling.6 SYBR Gold stain's other advantages include staining of RNA in many kinds of denaturing gels and the ability to penetrate thick gels. It also has a peak excitation at about 300 nm, which more closely matches the standard 302 nm UV transilluminators, allowing the researcher to get extremely high sensitivity without using expensive laser scanners. SYBR Green II stain is also available for staining RNA. While most dyes exhibit a greater fluorescence enhancement upon binding DNA than RNA, SYBR Green II stain has an RNA-bound quantum yield that is ~1.5 times greater than its DNA-bound yield. Under optimal staining and illumination conditions, SYBR Green II stain can detect as little as 100 pg of rRNA. SYBR Gold stain is more sensitive but does not enhance RNA signals relative to DNA. It is possible to add SYBR dyes to cast gels or to running buffer, but some sources claim this reduces sensitivity. SYBR dyes do affect DNA mobilities. These dyes are generally supplied as a 10,000x stock in DMSO, and need to be diluted shortly (<24 hours) before use. Optimal staining is achieved in clear polypropylene containers as the SYBR dyes have been reported to bind glass and certain other plastics. Staining dishes should be rinsed with water only (no detergents) and used exclusively for SYBR dyes. Other dyes that outperform EtBr can be found as well. BioWhittaker Molecular Applications of Rockland, Maine, makes GelStar®, which can detect 20 pg of dsDNA or 10 ng of RNA in sharp bands. GelStar is a good stain for thick or high-percentage agarose gels and has low intrinsic fluorescence, eliminating the need for destaining. Bio-Rad of Hercules, Calif., offers Radiant Red, an RNA stain that emits a visible red-orange fluorescence under 302 nm excitation. Radiant Red performs well under denaturing conditions and can be used to stain formaldehyde and glyoxal gels with no pretreatment. A novel twist on gel staining comes from dyes such as Nile Blue. This stain can be added to the gel or running buffer at about 1 µg/ml. Unlike EtBr, the dye:DNA complex is visible with no special illumination, so you can watch the bands resolving as the gel runs.7 Nile Blue allows visualization of bands containing as little as 40 ng of DNA under ambient lighting. Nile Blue is essentially nontoxic, so it's a great reagent for schools and other labs in which equipment is minimal or safety is paramount. Further, enzymatic reactions can be performed on Nile Blue-stained DNA without removing the dye. Other visible dyes, such as amido black or CarolinaBLU™ from Carolina Biological Supply Co. of Burlington, N.C., can also be used for DNA staining. According to Carolina Biological, CarolinaBLU allows visualization of bands containing as little as 0.2 µg under ambient lighting, but a white light box or similar light source enhances visualization of bands. Solution Solutions Nucleic acids absorb UV light at 260 nm; a solution of pure dsDNA that has an optical density (OD) of 1.0 has a concentration of 50 µg/ml; an OD of 1.0 for ssDNA is about 33 µg/ml; and an OD of 1.0 for pure RNA is 40 µg/ml. But there are problems with using absorption to estimate nucleic acid concentration. The technique cannot discriminate between DNA, RNA, and free nucleotides and is affected by other UV-absorbing contaminants such as proteins in the sample. Further, since optical absorbance is a relatively insensitive technique, measurements consume an enormous amount of material. DNA has intrinsically low fluorescence, but measuring the fluorescence of a dye that specifically binds nucleic acids eliminates many of these problems. EtBr has been used, but other dyes such as Hoescht 33342 and Hoescht 33258 have seen more extensive use for quantifying DNA in solution. The method is simple: A sample of diluted dye is used to zero a fluorescence spectrometer. 1 µl of a solution of DNA of known concentration (often 0.1 mg/ml) is added and the fluorescence measured. The ratio of the fluorescence of 1 µl of test DNA in the same volume of diluted dye to the fluorescence of the standard allows calculation of the test DNA concentration. In addition to fluorometers, several other less expensive instruments are available for simple calibration and direct readout of DNA concentration. Because the technique uses only 1 µl of each DNA sample to be tested, it can be used to accurately determine the concentration of miniprep DNA, sequencing templates, and some PCR targets or products. Depending on the instrumentation, fluorometry using Hoescht 33258 is accurate down to ~10 ng/ml (not much better than EtBr), but it has the advantage of being specific for DNA. This is because it binds A/T base pairs, which are only found in dsDNA and not RNA. Most protocols use calf thymus DNA for the standard solution, which has an A/T content of 58 percent, so if your target DNA differs greatly from this, a different standard should be used. The unsymmetrical cyanine dyes have very low intrinsic fluorescence quantum yields (typically 1,000-fold) upon binding DNA.8 With odd names like POPO™, BOBO™, TOTO®, and YOYO®, these dyes offered by Molecular Probes form the basis for a number of sensitive assays with detection limits down to ~2.5 ng/ml.9 A number of unsymmetrical cyanine dyes have subsequently been released that are specific for particular applications and have exquisite sensitivity. Molecular Probes' PicoGreen® reagent is a fluorescent dye for quantifying dsDNA in solution. PicoGreen reagent quantifies as little as 25 pg/ml in a conventional fluorometer cuvette. Microtiter-based assays are generally far less sensitive due to small volumes and increased background fluorescence from the plastic; a limit of 250 pg/ml is cited for PicoGreen reagent in this format. PicoGreen reagent is not specific for dsDNA but the enhancement of fluorescence is greater on binding dsDNA than on binding RNA or protein, allowing dsDNA to be quantified in the presence of contaminating RNA or protein. The PicoGreen assay is many times more sensitive than Hoescht 33258, and as it exhibits much less base selectivity, it can accurately quantify DNA from a wider range of sources. Molecular Probes' OliGreen® reagent is a variant that is optimized for ssDNA and synthetic oligonucleotides. Oligos as short as 20 nucleotides can be quantified with a detection limit of ~100 pg/ml in a standard fluorescence cuvette. RiboGreen® reagent is another variant, optimized for RNA detection. RiboGreen reagent detects 1 ng/ml of RNA in a standard fluorometer. RiboGreen reagent detects DNA as well, but can be used for an RNA-specific assay by simply pretreating the samples with DNase. These reagents are generally unaffected by the presence of common contaminants to DNA and RNA preparations such as proteins or free nucleotides, contaminants that can significantly influence the accuracy of traditional absorbance-based methods. PicoGreen and OliGreen reagents also have a linear dynamic range of four orders of magnitude with a single dye concentration, making it easy to automate assays for high-throughput applications or any application in which the nucleic acid concentration is completely unknown. RiboGreen reagent covers three orders of magnitude with two dye concentrations. With availability of the many alternative nucleic acid dyes mentioned above, ethidium bromide may become one of the dusty relics on the chemical shelf serving only as a reminder of days past. Bob Sinclair can be contacted at bobsinclair@tech-write-edit.com. References 1. P.A. Sharp et al., "Detection of two restriction endonuclease activities in Haemophilus parainfluenzae using analytical agarose-ethidium bromide electrophoresis," Biochemistry, 12:3055-63, 1973. 2. G. Lunn, E.B. Sansone, "Ethidium bromide: destruction and decontamination of solutions," Analytical Biochemistry, 162:453-8, 1987. 3. J. Sambrook et al., Molecular Cloning: A Laboratory Manual, New York, Cold Spring Harbor Laboratory, 1989. 4. P. Quillardet, M. Hofnung, "Ethidium bromide and safety--readers suggest alternative solutions," Trends in Genetics, 4:89, 1988. 5. V.L.Singer et al., "Comparison of SYBR Green I nucleic acid gel stain mutagenicity and ethidium bromide mutagenicity in the Salmonella/mammalian microsome reverse mutation assay (Ames test)," Mutatation Research, 439:37-47, Feb. 2, 1999. 6. R.S. Tuma et al., "Characterization of SYBR Gold nucleic acid gel stain: a dye optimized for use with 300-nm ultraviolet transilluminators," Analytical Biochemistry, 268:278-88, March 15, 1999. 7. S. Adkins, M. Burmeister, "Visualization of DNA in agarose gels as migrating colored bands: applications for preparative gels and educational demonstrations," Analytical Biochemistry, 240:17-23, 1996. 8. V.L. Singer et al., "Characterization of PicoGreen reagent and development of a fluorescence-based solution assay for double-stranded DNA quantitation," Analytical Biochemistry, 249:228-38, 1997. 9. H.S. Rye et al., "Fluorometric assay using dimeric dyes for double- and single-stranded DNA and RNA with picogram sensitivity," Analytical Biochemistry, 208:144-50, 1993.

An Eye for a Dye

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