Methylene blue : an alternative , multi-purpose stain for detection , analysis and isolation of nucleic acids

A series of experiments was performed utilizing Methylene Blue (MeB) in place of the intercalating dyes ethidium bromide (EtBr) and acridine orange (AO) to stain, visualize, and isolate DNA and RNA. MeB proved to be superior to the other dyes for several purposes: 1) visualization of glyoxalated (chemically denatured) RNA in agarose gels, 2) staining of nucleic acids that are to be used in subsequent hybridization experiments, and 3) isolation and purification of plasmid DNA by CsCl ultracentrifugation. MeB was found to perform at least as well as EtBr or AO for visualization of DNA in agarose of acrylamide gels, and DNA stained with MeB can be purified from agarose gel slices by the Gene Clean protocol. These results indicate that MeB is a very effective nucleic acid stain. Its safety versus conventional intercalating dyes wilt be discussed.


A series of experiments
was performed utilizing Methylene Blue (MeB) in place of the intercalating dyes ethidium bromide (EtBr) and acridine orange (AO) to stain, visualize, and isolate DNA and RNA.MeB proved to be superior to the other dyes for several purposes: 1) visualization of glyoxalated (chemically denatured) RNA in agarose gels, 2) staining of nucleic acids that are to be used in subsequent hybridization experiments, and 3) isolation and purification of plasmid DNA by CsCl ultracentrifugation.MeB was found to perform at least as well as EtBr or AO for visualization of DNA in agarose of acrylamide gels, and DNA stained with MeB can be purified from agarose gel slices by the Gene Clean protocol.
These results indicate that MeB is a very effective nucleic acid stain.Its safety versus conventional intercalating dyes wilt be discussed.
Introduction.Detection and analysis of nucleic acids is an integral part of molecular biology, and several dyes are commonly used for this purpose, including ethidium bromide (EtBr) and acridine orange (AO).The ideal stain is rapid, sensitive, stable, non-toxic, required no special equipment for use, is useful for double and single stranded nucleic acids, and does not interfere with subsequent hybridization expe riments.We have investigated the use of methylene blue (MeB) as an alternative to EtBr and AO, and found it to perform as well as or better than these intercalating dyes in many experimental procedures.Additional advantages to the use of MeB are that exposure to ultra-violet (UV) light is not necessary to visualize DNA or RNA, thus eliminating undesirable UV cross linking of samples and exposure of labo ratory personnel to the potentially harmful effects of UV light.Evidence also indicates that MeB is less toxic than either EtBr or AO, making it safer to use and easier to dispose of than standard intercalating dyes.The lowest reported lethal dose (LDLo) for intra peritoneal administration in mice is 20 mg/kg for
Materials and Methods.Methylene blue staining of denatured RNA.Duplicate sets of samples, con sisting of 15 jug total soybean leaf RNA, were electrophoresed in identical 1.0 % (w/v) agarose gels in 10 mM sodium phosphate buffer, pH 7.0.RNA was visualized by one of two methods: 1) stained in 10 ^g/ml MeB in DEPC-treated, sterile water for 15 min, then destained in 3 changes of deionized water over approximately 1 hr, or 2) stained in 1 jug/ml AO in sterile water, then destained.
Densitometer scans of autoradiographs were per formed on an LKB.Ultroscan XXL Laser Den sitometer with LKB 2400 Gelscan XL software version 1.0.Peak areas were calculated as: peak area = mm * AU h33 nm .
The valley-to-valley method was used to calculate baselines, and data were normalized to the strongest signal.

Use of methylene blue in DNA isolation and recovery.
Standard large scale preparations of pUCI 18/119 plasmid DNA were prepared by the alkaline lysis method [4].The density of the crude plasmid DNA solutions was adjusted to 1.55 g/ml with solid CsCI, and equal volumes (6.2 ml) of this solution were transferred to 2 15-ml Corcx tubes.300 //•I of 10 mg/ml EtBr or 300 p\ of 2 mg/ml MeB was added, the density readjusted to 1.55 g CsCl/ml, and tubes were centrifuged for 5 min at 7000*g to pellet remaining precipitated proteins and cell debris.The supernatant fractions were transferred to 5-ml Beckman quick-seal tubes (the pellet in the MeB tube was looser and more flaky than that in the EtBr tube, making it more difficult to avoid transferring some solid to the quick-seal tube).
U1 tracentrifLigation was performed at 50,000*g for about 15 hr at 20 C. Plasmid DNA bands were clearly visible in each tube, and were extracted with a 16 gauge needle attached to a 3-mI syringe.100/її more of each stain was added to the appropriate sample, and a second round of ultracentrifugation was performed as above.Distinct plasmid bands were visible in both tubes (Fig. 4); note that MeB-stained plasmid DNA migrates further down the CsCI gra dient due to its increased density.Plasmid DNA bands were extracted from the tubes as above./т-Butanol was found to extract MeB and EtBr equally well from the samples (data not shown).Following dialysis in ТЕ buffer pH 8.0 (ТЕ -10 mM Tris, 1 mM EDTA) to remove remaining CsCI |4], DNA samples were precipitated with 95 % (v/v) ethanol, dried under vacuum, and resuspended in sterile ТЕ.Concentrations were determined speetrophotomctrically on duplicate samples.
Results and Discussion.Although methylene blue is commonly used as a stain for the detection of RNA on Northern blots [5,6 ], it has not been tested as a general purpose nucleic acid stain.Thus, we sub stituted MeB for traditional intercalating dyes, ethi dium bromide and acridine orange, in several mole cular biology procedures.Use of MeB was found to be preferable to intercalating dyes for staining glyoxalated RNA in agarose gels (Fig. 1).Glyoxal che mically denatures RNA by covalently modifying gua nine residues, and glyoxalated RNA does not stain well with EtBr or AO unless the glyoxal is first removed from the gel by brief treatment with 0.05 M NaOH and neutralization [ For visualizing DNA after electrophoresis, MeB provides comparable sensitivity to EtBr in agarose gels (Fig. 3), and in acrylamide gels (data not shown).MeB-stained gels can be viewed in ambient room light and photographed on a white light box; they require no exposure of the investigator or DNA samples to UV light.In contrast to EtBr, however, MeB can not be used in the gel to stain nucleic acids during electrophoresis, as this was found to cause distortions in the migration of DNA fragments.
MeB can also be substituted for traditional inter calating dyes in the isolation and purification of plasmid DNA by CsCl density ultracentrifugation (Fig. 4).Surprisingly, plasmid yield after CsCl den- Ethidium Bromide Methylene Blue Fig. 3. Visualization of f/indllf restriction endonuolease-digcste( lambda DNA after electrophoresis.Duplicate sets of 500, 100, 50 and 10 ng samples (lanes / through 4, respectively) were elec trophoresed and stained with either 1 /tg/ml EtBr or \0 ug/m\ MeB MeB stained the DNA at least as effectively as the more common!;used dye, EtBr sity ultracentrifugation with MeB staining was up to 10-fold higher than with EtBr staining.While we cannot explain this result, the increased yield with MeB was consistent and repeatable.Both MeB and EtBr plasmid preparations yielded very pure DNA that was easily digestible with restriction endonuc-Ieases.
Finally, we determined that agarose gel slices containing MeB-stained DNA bands could be pro cessed by the Gene Clean protocol (Biol01, La Jolla, CA) to recover DNA fragments for cloning.DNA fragments stained with either EtBr or MeB are equally recoverable by this procedure (data not shown).
Tn this safety-conscious age, it is desirable to develop molecular biology protocols that are less hazardous to the scientist and have fewer negative environmental side effects than conventional methods.This series of experiments was carried out to demon strate that MeB can be used in place of the more hazardous intercalating dyes EtBr or AO in many applications involving DNA or RNA.
Acknowledgments.We thank L. Todd Buckman for his invaluable help with the photography and dark room work.

Fig. 4 .
Fig. 4. Cesium chloride density gradient purification of plasmid DNA.Crude plasmid DNA was prepared by a standard alkaline lysis protocol and then purified by ultracentrifugation in a CsCl gradient, utilizing either FvtRr or MeB for visualization of the plasmid band (see «Materials and Methods» for details).Arrows indicate the position of each band; note that MeB increases the density of the DNA, resulting in its migration further down the tube 7|, which we found to have undesirable effects on RNA stability and its transfer Because visu alization of RNA by MeB staining does not require prior removal of glyoxal from the gel, denatured RNA can be processed as the glyoxalated adduct throu ghout electrophoresis and transfer, thus minimizing the risk of RNA degradation [8 |.
Fig.1.Visualization of total RNA isolated from the soybean cultivars Acme (A) and Norchief (AO.Fifteen ug samples of glyoxalated (denatured) RNA were electrophorcsed as described in «Materia Is and Mcthods» and stained with 1 /*g/ml AO or 10wg/ml MeB.MeB was more effective than intercalating dyes for staining chemically denatured RNA to nylon membranes.Tn addition, our results indicate that binding of a u P-labeled cDNA probe to AOstained RNA samples was inhibited about two-fold in comparison to McB-stained samples (Fig.2), de monstrating that MeB-staincd RNA is also superior for hybridization to DNA probes.