Proc. Natl. Acad. Scf. USAVol. 74, No. 11, pp. 4835-4838, November 1977Biochemistry

Analysis of single- and double-stranded nucleic acidson polyacrylamide and agarose gels by using glyoxaland acridine orange

(RNA/DNA/denaturation/molecular weights/gel electrophoresis)

GARY K. MCMASTER* AND GORDON G. CARMICHAELtt* Unit of Developmental Biology and t Department of Virology, Swiss Institute for Experimental Cancer Research, Chemin des Boveresses, 1066 Epalinges,Lausanne, Switzerland

Communicated by Walter Gilbert, September 8, 1977

ABSTRACT We have developed a simple and rapid systemfor the denaturation of nucleic acids and their subsequentanalysis by gel electrophoresis. RNA and DNA are denaturedin 1 M glyoxal (ethanedial) and 50% (vol/vol) dimethyl sulfoxide,at 500. The glyoxalated nucleic acids are then subjected toelectrophoresis through either acrylamide or agarose gels in a10 mM sodium phosphate buffer at pH 7.0. When glyoxalatedDNA molecules of known molecular weights are used as stan-dards, accurate molecular weights for RNA are obtained. Fur-thermore, we have employed the metachromatic stain acridineorange for visualization of nucleic acids in gels. This dye in-teracts differently with double- and single-stranded polynu-cleotides, fluorescing green and red, respectively. By using thesetechniques, native and denatured DNA and RNA molecules canbe analyzed on the same slab gel.

The electrophoretic mobility of nucleic acids in polyacrylamideor agarose gels depends on both molecular weight and confor-mation (1, 2). Removing secondary and tertiary structure shouldmake the electrophoretic mobility a simple function of mo-lecular weight. Gels containing the denaturing agents formal-dehyde (3), formamide (4,5), methylmercuric hydroxide (6),and urea (7) have all been used for molecular weight determi-nations. Here we present a simple and convenient method thatwe feel offers a number of advantages over those previouslydescribed. This method employs denaturation of nucleic acidsand reaction with glyoxal, followed by electrophoresis in a slabgel.

Glyoxal (ethanedial) reacts with nucleic acids, nucleotides,and their component bases (8). Although at high concentrationsglyoxal has been reported to react with all bases of both RNAand DNA (8), the guanosine-glyoxal adduct is by far the moststable (8-10). Glyoxalation introduces an additional ring ontoguanosine residues, thus sterically hindering the formation ofG-C base pairs and consequently the renaturation of nativestructure (11). Previously, glyoxalation has been used as a toolfor the denaturation of DNA and RNA for visualization byelectron microscopy (12-16).

Ethidium bromide is commonly used as a sensitive stain fordetecting nucleic acids in gels (17, 18). However; this dye cannotdistinguish between single- and double-stranded polynucleo-tides. Acridine orange has been employed extensively as a cy-tochemical stain (for review, see ref. 19) and h ' -- chownto stain differentially DNA and RNA, and double-.,. rle-stranded nucleic acids in situ (20, 21). Acridine orange caneither intercalate into double helical nucleic acids (green flu-orescence at 530 nm) (22, 23), or bind electrostatically tophosphate groups of single-stranded molecules (red fluorescence

at 640 nm) (24, 25). We have exploited this property to staindifferentially single-stranded (red) and double-stranded (green)polynucleotides in gels.

MATERIALS AND METHODS

Materials. From Fluka (Buichs, Switzerland) we obtainedacridine orange (standard for microscopy), glyoxal (technicalgrade, 30% (wt/vol) solution in H20], dimethyl sulfoxide(Me2SO, analytical grade), acrylamide (practical), N,N'-methylenebisacrylamide (practical), and N,N,N',N'-tetra-methylethylenediamine. For best results with RNA the acryl-amide and N,N'-methylenebisacrylamide were recrystallizedaccording to Loening (1). Mixed bed ion-exchange resin, AG-501-X8 or AG-501-X8(D), was from Bio-Rad, and agarose,electrophoresis grade, was from Sigma. All other chemicals usedwere reagent grade, and before use buffers were filteredthrough Millipore nitrocellulose membrane filters.

Glyoxal Purification. Because glyoxal is readily air-oxidized,we remove oxidation products the same day as the samples areto be prepared. This is accomplished by passing the glyoxalsolution three times through a column (10-ml pipette) con-taining mixed bed resin. A column volume of 1 ml is sufficientto remove the oxidation products from 2 ml of 3 glyoxal. Thisglyoxal purification step is mandatory for the prevention ofRNA fragmentation.RNA and DNA Samples. The following ribosomal RNAs

were used in these studies: Escherichia coli (5S, 16S, 23S, fromP. Wellauer, or phenol extracted from strain K-12 by ourselves),HeLa (18S, 28S, from P. Wellauer), mouse mastocyte cells (18S,28S, from D. Hughes), Physarum polycephalum (19S, 26S,from U. Guebler and R. Braun). In addition, we used E. coli 4SRNA (from P. Wellauer) and duck globin mRNA (from G.Spohr).

Simian virus 40 (SV40) closed circular viral DNA was pre-pared from virus-infected monkey cells as described by others(26). Restriction endonucleases EcoRI (Escherichia coli RI),BamHI (Bacillus amyloliquefaciens), Hpa II (Haemophilusparainfluenzae), and HindIII (Haemophilus influenzae d)were purchased from New England Bio Labs, Beverly, MA, andused as suggested by the supplier. Each of the restriction en-donucleases EcoRI, BamHI, and Hpa III cuts the SV40 genomeat a unique site (17, 27, 28). Cleavage with EcoRI generatesfull-length, double-stranded linear molecules (27). Cleavagewith a mixture of BamHI and Hpa II yields two fragments, ofapproximately 57% and 43% unit length (28). We calculate themolecular weights of these fragments to be 1.92 X 106 and 1.46

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby marked.advertasement" in accordance with 18 U. S. C. §1734 solely to indicatethis fact.

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Abbreviations: Me2SO, dimethyl sulfoxide; SV40, simian virus 40.* Present address: Department of Pathology, Harvard Medical School,25 Shattuck Street, Boston, MA 02115.

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X 106, respectively; full-length SV40 DNA is about 5200 basepairs, known from recent sequence studies (see ref. 28 for re-view) (molecular weight = 3.4 X 106, assuming an average basepair has a molecular weight of 660). HindIll cuts SV40 DNAinto six fragments (29). In order of decreasing size, the HindIIIA-F fragments are 34%, 22.5%, 20.5%, 10.5%, 8.5%, and 4.0%unit length (29). In size they range from 1.16 X 106 daltons(fragment A) to 0.136 X 106 daltons (fragment F). Before de-naturation and glyoxalation, all DNA samples were depro-teinized by phenol extraction and concentrated by ethanolprecipitation.Sample Denaturation. RNA and DNA samples to be dena-

tured are incubated in capped plastic tubes for 1 hr at 50° ina buffer containing 1.0 M glyoxal, 50% (vol/vol) Me2SO and10 mM NaH2PO4/Na2HPO4, pH 7.0. Control samples aretreated identically, but without glyoxal and Me2SO. After in-cubation the samples are cooled to room temperature and su-crose is added to 5% just before electrophoresis.We have investigated the effects of glyoxal concentrations

from 10 mM to 2.0 M and Me2SO concentrations from 0 to 80%on a large number of nucleic acids. Samples have been incu-bated at temperatures from 200 to 70°. Glyoxal denaturationlowers the electrophoretic mobility of nucleic acids. By thiscriterion of lowered mobility, the sample denaturation schemepresented above has proven sufficient for complete denatura-tion (maximum decrease in mobility) of all samples we havetested. Denaturation of RNA under the same conditions, butwithout glyoxal, allows renaturation and has not altered themobilities of RNAs we have tested. Furthermore, addingglyoxal to the gel buffer has not altered detectably the mobilitiesof nucleic acids already completely denatured with this reagent.We have stored glyoxalated samples for 2 days at 40 and havenoticed no degradation of either DNA or RNA.

Polyacrylamide/Agarose and Agarose Slab Gels. Poly-acrylamide/agarose composite slab gels are according to Pea-cock and Dingman (30), using 10mM sodium phosphate buffer,pH 7.0. Composite gels contain 0.5% agarose and 1.5-2.5%polyacrylamide. We have also used 1% or 1.5% agarose slab gelscontaining sodium phosphate buffer as above in either a hori-zontal (31) or a vertical gel apparatus. Specific electrophoresisconditions are in Table 2. During electrophoresis it is importantto recirculate the low ionic strength buffer; for long electro-phoresis times the buffer should be changed every 2.5 hr.Current should never exceed 45 mA. Slab gels are 2.5-3 mmthick and are 10 cm long.

Gel Staining. Polyacrylamide/agarose composite gels arestained in acridine orange at 30 ,gg/ml for 15 min in 10 mMphosphate buffer, pH 7.0. Agarose gels are stained for 30 min.The gels are then transferred to a flat, enameled metal pan fordestaining in the same buffer. Enamel adsorbs acridine orange,thereby reducing the time required to remove the high back-ground fluorescence of the gel. The stain is removed from thepan by rinsing with hot running tap water for 5-10 min. Poly-acrylamide-containing gels are destained in the dark for 2 hrat 220, or at 40 overnight. Agarose gels are sufficiently destainedafter 1 hr at 220. Using acridine orange, one can easily detect,by UV illumination, 0.05 mg of double-stranded and 0.1 ,g ofsingle-stranded polynucleotide in a gel band. We have observedthat ethidium bromide is much less sensitive than acridine or-ange for the detection of single-stranded glyoxalated nucleicacids: glyoxal may react with ethidium, changing its spectralproperties (14).

Photography. The destained gel is placed on a UV transil-luminator (Xmax = 254 nm) (model C-61, Ultraviolet Products,San Gabriel, CA). Color photography is with Polaroid type 108color film and a yellow filter. Black and white photography is

FIG. 1. Native and glyoxal-denatured nucleic acids were elec-trophoresed on one gel and stained with acridine orange. Migrationis toward the anode, at the bottom. Restriction nuclease fragmentsof SV40 DNA and various rRNA species were electrophoresed 4 hrat 130 V through a 2% polyacrylamide/0.5% agarose gel, either nativeor after denaturation with Me2SO and glyoxal. The gel was stainedwith acridine orange and destained as described in Materials andMethods. (a) 1 pg of E. coli 16S rRNA, denatured. (b) 0.4 pg of full-length linear SV40 DNA formed by EcoRI digestion of viral DNA,not denatured. Upper band is nicked circular DNA, from incompletedigestion. (c) 0.8 pg of SV40 full-length linear DNA, denatured. (d)0.8 pg of SV40 DNA fragments (A-D) formed by HindIII digestionof viral DNA, not denatured. Fragments E and F have run off thebottom of the gel, and the upper band is full-length linear DNA fromincomplete digestion. (e) 2 pg of SV40 HindlIl fragments A-E, de-natured. (f) 1 jg of E. coli 23S rRNA, not denatured. Upper greenband is high molecular weight DNA contaminant. (g) 1 pg of E. coli23S rRNA, denatured. (h) 1 pg of Physarum polycephalum 26SrRNA, denatured. (i) 1 pg of 28S mouse rRNA, not denatured. () 1pg of 28S mouse rRNA, denatured.

with Polaroid type 105 positive/negative film or type 107cpositive film and a red filter.

RESULTSRNA and DNA molecules from various sources were denaturedwith Me2SO and glyoxal, electrophoresed along with unde-natured samples in the same slab gel, and detected by acridineorange staining. Fig. 1 shows the analysis of both native anddenatured DNA and RNA on a 2% polyacrylamide/0.5%agarose composite slab gel. Fig. lb shows by green fluorescencefull-length double stranded linear SV40 DNA molecules formedby cleavage of the circular viral genome with the restrictionendonuclease EcoRI. When the same material is denatured inglyoxal and Me2SO, the full-length linear single strands migratemore slowly and fluoresce red (1c). Single-stranded SV40 DNAcircles, when glyoxalated, migrate more rapidly than linearmolecules (1c, faint band), perhaps because of the physicalconstraints on complete unfolding imposed by the circularityof the molecule. Fig. ld shovs native SV40 DNA fragments(green) formed by digestion of the viral DNA with the re-

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FIG. 2. Glyoxal-denatured nucleic acids electrophoresed in ahorizontal 1% agarose gel. Samples were denatured with Me2SO andglyoxal and electrophoresed 2 hr at 100 V (Na-PO4 buffer). All bandsappeared red after acridine orange staining. (a) 1 Mg of 16S E. colirRNA. (b) 0.4 jtg ofSV40 linear DNA. (c) 2 fig ofSV40 DNA partiallydigested with Hpa 11+ BamHI. The most prominent band is unitlength. The arrows point to the fragments of 57% and 43% unit length.(d) 2 jig of SV40 HindIII DNA fragments A, B, and C. Fragments Eand F have run offthe bottom ofthe gel; B and C are not resolved here,due to overloading. (e) 2 Mig of 28S mouse rRNA. (f) 1 Mg of 26S Phy-sarum polycephalum rRTNA. (g) 1 Mig of 23S E. coli rRNA. Upper bandis high molecular weight DNA. (h) 1 Mg of 19S Physarum poly-cephalum rRNA, slightly contaminated with 26S rRNA. (i) 1 Mg of18S mouse rRNA. (j) 1 ug of 16S E. coli rRNA.

striction endonuclease HindIII. When denatured, these frag-ments fluoresce red and the migration is again decreased(le).RNAs we have used are primarily single-stranded, fluores-

cing red under all conditions. Denaturation and glyoxalationcauses a striking reduction in their electrophoretic mobility,perhaps reflecting the disruption of extensive secondarystructure. For example, compare native and denatured E. coli23S rRNA (if, red band vs. lg) and mouse 28S rRNA (Ii vs. ij).The 23S rRNA preparation contains high molecular weightbacterial DNA (if, green band). The green band disappearedafter DNase treatment, while the red band disappeared afterRNase treatment (data not shown). Fig. la shows the migrationon this gel of denatured E. coli 16S rRNA, and lh shows themigration of denatured 26S rRNA of Physarum polycepha-lum.The glyoxal method can also be used to estimate molecular

weights of single-stranded nucleic acids. Fig. 2 shows a varietyof glyoxal-denatured DNA and RNA molecules that have beenelectrophoresed in a horizontal 1% agarose gel. When the rel-ative mobilities of the denatured DNAs (restriction endonul-cease fragments of SV40 DNA) are plotted as a function of theirknown molecular weights, on a logarithmic scale, a straight lineis obtained (Fig. 3). Interestingly, if we place the mobilities ofthe denatured RNA species on this same line, we can interpolateRNA molecular weights that are in excellent agreement withpublished values (Table 1). We have observed this phenomenonafter electrophoresing glyoxal-denatured nucleic acids in a largenumber of gel systems, all using low ionic strength phosphatebuffer. Furthermore, by choosing the appropriate gel system,one may estimate RNA molecular weights over a very wide

5.0 6.0 7.0 8.0 9.0 10.0Relative mobility, arbitrary units

FIG. 3. Semi-logarithmic plot of relative electrophoretic mobilityvs. molecular weight. Relative mobilities of the denatured RNA andDNA species in Fig. 2 were measured as the distances from the centerbottom of the sample wells to the center top ofthe bands. When bandintensities are not the same, this method is more accurate (comparefor example, two different amounts of full-length SV40 DNA, shownin Fig. 2 b and c). When bands are ofequal intensity, equivalent resultsare obtained by measuring to the center of the bands. The Os repre-sent SV40 DNA fragments produced by restriction enzyme cleavage.The line has been drawn through them. The *s represent rRNAs andhave been placed on the line in order to interpolate molecularweights.

range (Table 2). Table 2 also shows that glyoxal-denatured DNAand RNA molecules have the same mobilities over a 5-foldrange of molecular weights, on the same gel.

DISCUSSIONWe have described here a combination of two useful techniquesfor the analysis of nucleic acids by gel electrophoresis; glyoxaldenaturation and discrimination of double-stranded (green)and single-stranded (red) polynucleotides by acridine orangestaining. These techniques may be used independently or inconjunction.With glyoxal, nucleic acids are essentially irreversibly de-

natured under the conditions we use for sample preparation andgel electrophoresis. At pH 7, the glyoxal-guanosine adduct is

Table 1. Comparison of published estimates for RNA molecularweights with those determined by the glyoxal method.

Molecular weight X 10-6RNA Glyoxal gel Published Refs.

16S E. coli 0.55 + 0.03 0.54 + 0.02 33, 3423S E. coli 1.03 0.03 1.07 3418S Mouse 0.68 1 0.02 0.68 b 0.06 3528S Mouse 1.75 ± 0.05 1.74 b 0.1 3518S HeLa 0.67 + 0.02 0.68 + 0.07 36, 3728S HeLa 1.75 + 0.05 1.76 + 0.15 36, 3719S Physarum 0.70 + 0.02 0.76 + 0.01* 3826S Physarum 1.29 b 0.03 1.37 ± 0.05* 389S Duck globin

mRNA 0.21 + 0.02 0.205 ± 0.02 5

RNA molecular weights were determined after gel electrophoresisfrom standard curves derived using denatured SV40DNA restrictionendonuclease fragments ofknown size (28,32). Molecular weights areexpressed as the sodium salt of the polynucleotide. Each value shownreflects the average of at least five separate determinations usingdifferent gel systems.* Determined under nondenaturing conditions.

28S Mouse

1 00 26S Physarum

23S E. coli

57% 19S PhysarumMouse

4_ 1 6S E. co/i34%

22.5%

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Table 2. Gels used for molecular weight estimations

Running Approximateconditions "linear range,"

Gel* Min V X 10-6 t

2.5% composite 105 130 0.025-0.20 (4S-9S)2.5% composite 180 130 0.20-0.70 (9S-18S)2.5% composite 300 130 0.50-1.4 (16S-26S)2.0% composite 300 130 0.50-1.75 (16S-28S)1.5% agarose 120 100 0.20-1.01.0% agarose

horizontal 120 100 0.40-1.75

* Composite gels always contained 0.5% agarose and the indicatedpercentage of acrylamide.

t Values are expressed as molecular weight, with sedimentation valuesin parentheses. Within this "linear range," the relative mobilitiesof glyoxal-denatured nucleic acids are directly proportional to thelogarithms of their molecular weights.

stable for at least 20 hr at 20° (9). This means that the gel orrunning buffers do not have to contain denaturing agents, thusmaking it possible to analyze both native and fully denaturedmolecules on the same slab gel. In addition, glyoxal-denaturednucleic acids may be electrophoresed through either agaroseor acrylamide gels. We have never seen evidence of intermo-lecular crosslinking induced by glyoxal, using RNA concen-trations from 1 to 800 ,sg/ml, and glyoxal concentrations from10 mM to 1.5 M. This is in agreement with physical chemicalstudies (11) and with electron microscope observations (P.Wellauer, personal communication).An interesting finding was that glyoxal-denatured RNAs and

DNAs fall on the same log molecular weight vs. relative mo-bility line, or on very similar lines. This means that it is possibleto estimate RNA molecular weights with the glyoxal methodby using easy-to-obtain DNA fragments of known size asmarkers. The RNA molecular weight values we have obtaineddo not seem to be highly dependent on the G+C content of theRNAs; molecules we have used vary in G+C content betweenabout 53% (16S E. coli) and about 67% (28S HeLa) (39). Thissystem is of further interest because formation of the glyoxal-guanosine adduct is readily reversible at pH greater than 8 (9).Shapiro et al. (40) described the complete dissociation of theglyoxal-guanosine adduct in 75 min at pH 11, 22 , while Huttonand Wetmur (11) reported the complete dissociation of glyoxalfrom DNA by treatment for 76 hr at pH 8.0, 450. Thus, theglyoxal method may be of use when the recovery of unglyox-alated nucleic acids is desired.The glyoxal method is simple and rapid. It requires relatively

safe, inexpensive reagents, may be adapted to any commonlyused gel apparatus, and is compatible with both acrylamide andagarose, and with both DNA and RNA. Thus, we feel that thismethod is in many respects superior to previously describedmethods for gel analysis of denatured DNA or RNA (3-7,31).We are grateful to all those who so generously supplied materials

used in this research. We thank Dr. David Appleby for help during theearly stages of this work, and Beatrice Bentele and Nadine Hauser fortechnical assistance. Nick Acheson, Gary Cohen, David Hughes,Yvonne Roth, Georges Spohr, and Peter Wellauer provided invaluablecomments during the course of this work and preparation of themanuscript. Special thanks go to Sohan Modak and Bernhard Hirt fortheir continued support and helpful criticisms on this manuscript. Thisresearch was supported by grants 3.538.75 (to S. Modak) and 3.738.76(to B. Hirt) from the Swiss National Science Foundation. G.G.C. wassupported by a postdoctoral fellowship from The Jane Coffin ChildsMemorial Fund for Medical Research. This work constitutes part ofthe doctoral thesis of G.K.M.

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