JOURNAL OF BACTERIOLOGY, Sept. 1976, p. 1529-1537Copyright © 1976 American Society for Microbiology

Vol. 127, No. 3Printed in U.S.A.

Simple Agarose Gel Electrophoretic Method for theIdentification and Characterization of Plasmid

Deoxyribonucleic AcidJANE ALDRICH MEYERS, DAVID SANCHEZ, LYNN P. ELWELL, AND STANLEY FALKOW*Department of Microbiology, University of Washington School of Medicine, Seattle, Washington 98195

Received for publication 13 April 1976

Argarose gel electrophoresis may be employed effectively for the detection andpreliminary characterization of plasmid deoxyribonucleic acid (DNA) present inclinical isolates and laboratory strains of gram-negative microorganisms. Themethod is sensitive and does not require radioisotopes or ultracentrifugation.The estimation of plasmid mass from the extent of DNA migration in gelscompares favorably with results obtained by electron microscopy of plasmidDNA purified by equilibrium density centrifugation. The method has proved tobe a useful tool for survey work and the epidemiological investigation ofplasmiddissemination, as well as an important adjunct to the genetic analysis ofplasmids.

Currently, the standard procedure for isolat-ing and characterizing plasmid deoxyribonu-cleic acid (DNA) depends upon lysing host bac-terial cells and subsequently treating the lysateso that the smaller circular plasmid DNA mole-cules are separated from the relatively hugemass of chromosomal DNA (6, 8, 13). An inte-gral part of this procedure requires either thesedimentation of the plasmid DNA through analkaline or neutral sucrose gradient or equilib-rium density centrifugation with an intercalat-ing dye such as ethidium bromide. Cesiumchloride-ethidium bromide equilibrium densitycentrifugation requires an extensive period ofcentrifugation at high speed. The plasmid DNAmay often be visualized directly in such densitygradients by use of long-wave ultraviolet light(5), but if one wishes to determine the numberand size of plasmid species it is necessary toeither examine the plasmid DNA under anelectron microscope or sediment labeled DNAthrough a sucrose gradient. Whole lysates con-taining labeled DNA may be sedimented di-rectly in alkaline sucrose gradients to deter-mine the number and size of resident plasmidspecies within a bacterial strain (8), but a largenumber of fractions must be counted in a liquidscintillation spectrometer. Moreover, it is oftennecessary to include a differentially labeledstandard plasmid DNA of known molecularweight in sucrose gradients to calculate the sizeof any unknown plasmid species. Thus, currentprocedures for the isolation and superficialcharacterization of plasmid DNA in even a sin-gle strain can be relatively lengthy and costly,

and the methods available are not well suitedfor survey work.Our laboratory has been particularly inter-

ested in examining the plasmids present inclinical isolates of gram-negative bacteria (5,11, 18). It has been also necessary for us todetermine the number and molecular weight ofcovalently closed circular (CCC) plasmid DNAspecies in laboratory strains after their conju-gation with a clinical isolate. Consequently, wehave been searching for an inexpensive screen-ing method for the detection of plasmids andthe determination of their size that could beapplied to a wide variety of bacterial species.Recently, agarose gel electrophoresis has beenwidely employed in the analysis of restrictionendonuclease-generated fragments of plasmidand viral DNA (2, 14). The use ofthis methodol-ogy for the characterization of CCC DNA hasreceived little attention. Aaij and Borst (1) re-ported that the migration rates of purified bac-teriophage and mitrochondrial CCC DNAsranging from 3.4 x 106 to 10 x 106 daltons wererelated inversely to the logarithm of their massin 0.6% agarose gels. The migration propertiesofhigher-molecular-weight CCC DNA has beenoverlooked, however.

In the present paper we report the use ofagarose gel electrophoresis for the detectionand preliminary characterization of CCC plas-mid DNA present in clinical isolates and labo-ratory strains of gram-negative microorga-nisms. We have found the method to be suitablefor the detection and estimation of plasmidDNA molecular weight ranging from 0.6 x 106

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to 95 x 106 in partially purified whole-cell ly-sates.

MATERIALS AND METHODSBacterial strains and plasmids. The F- derivative

of Escherichia coli K-12 strain J5-3, called SF400[pro-22 met-63 (X)], was employed as a standardhost for plasmid species. E. coli K-12 strain 711[F-(y) lac-28 his-51 trp-30 proC23 Phe, Nalr] wasemployed as recipient of plasmid species in geneticcrosses. The molecular properties of the plasmidsRldrd19 (62 x 106 daltons), RP4 (34 x 106 daltons),Sa (25 x 106 daltons), RSF1030 (5.5 x 106 daltons),and RSF1010 Apll3 (8.7 x 106 daltons) were de-scribed previously (4, 7, 12). The 1.8 x 106-daltonColEl derivative pMB8 was isolated by M. Betlachand H. W. Boyer. Strain B41, toxigenic for calves,was described previously (17, 18). Clinical isolates oftoxigenic E. coli from cases of traveller's diarrheawere provided by E. Gangarosa, Center for DiseaseControl, Atlanta, Ga. Haemophilus influenzaestrains containing plasmid-linked ampicillin resist-ance were described by Elwell et al. (5) The Neis-seria gonorrhoeae isolates employed were also char-acterized previously (16).

Methods of DNA isolation. (i) Preparation ofcrude lysates for agarose gel electrophoresis. Plas-mid-containing strains were grown overnight in 30ml ofbrain heart infusion broth (Difco Laboratories,Detroit, Mich.) and harvested by centrifugation.The cells were suspended in 1.5 ml of 25% sucrose in10 mM tris(hydroxymethyl)aminomethane (Tris)-1mM ethylenediaminetetraacetate (EDTA), pH 8.0.Cleared lysates were then prepared by the sodiumdodecyl sulfate (SDS)-salt precipitation method de-scribed by Guerry et al. (10). The volume of thecleared lysate was doubled by the addition of dis-tilled water, 20 ,mg of ribonuclease per ml (bovinepancreas, type IA, Sigma Chemical Co., St. Louis,Mo.; 1 mg/ml in 50 mM sodium acetate [pH 5.0]heated for 10 min at 90°C) was added, and the lysatewas incubated at 370 for 1 h. After ribonucleic aciddigestion, 1 volume of Tris (50 mM)-saturatedphenol was added. The tube was inverted gentlyseveral times and centrifuged at 12,100 x g for 30min at 20°C in a refrigerated centrifuge to obtain aclear aqueous phase. Lysates from some clinical iso-lates required repeated rounds of phenol extractionand centrifugation to obtain a clear aqueous phase.When repeated phenol extractions were necessary,the loss of plasmid DNA was minimized by collect-ing the aqueous-phenol interface after each extrac-tion. The combined interface material was pooledand extracted once more with phenol and thisaqueous phase was added to the total aqueous vol-ume.The clear aqueous phase was brought to 0.3 M

sodium acetate (final concentration), and twice thevolume of cold (-20°C) 95% ethanol was added toprecipitate the DNA. The tube was kept at -200C for2 to 3 h (or overnight if more convenient). Theprecipitated DNA was recovered by centrifugationat 12,000 x g at - 10°C for 20 min. The ethanol wasthoroughly drained from the tube, and the DNA wassuspended in 100 ,lp of TES buffer (50 mM NaCl, 5

mM EDTA, 30 mM Tris, pH 8.0). The DNA samplewas analyzed immediately by agarose gel electro-phoresis or stored at -20°C until ready for use.

(ii) Preparation of large quantities of purifiedplasmid DNA. The polyethylene glycol (PEG) pre-cipitation method (15) was used to obtain largequantities of plasmid DNA contained in E. coli K-12or clinical isolates. The plasmid DNA was purifiedby cesium chloride-ethidium bromide equilibriumdensity centrifugation, and the purified plasmidDNA was collected by fractionation (4, 7). The ethid-ium bromide was removed by dialysis against TES.The purified DNA was stored at -20°C. Althoughplasmid DNA standards can be prepared individ-ually and mixed, we found it convenient to simplyco-cultivate different E. coli K-12 sublines contain-ing standard plasmid species, starting with an equalinoculum of each strain (106 cells per ml) into 500 mlof brain heart infusion broth.

It should also be noted that the PEG method canalso be employed as an alternative to the SDS-saltprocedure described above. Rather than using den-sity gradient centrifugation, one simply resuspendsthe PEG precipitate as obtained in this procedure ina small volume of 7 M CsCl. The PEG is insoluble atthis concentration, so that removal of the aqueousphase, followed by dialysis against TES, yields apartially purified plasmid preparation that may beanalyzed directly by gel electrophoresis.Agarose gel electrophoresis ofDNA. Ethanol-pre-

cipitated DNA (2 to 25 1l) from cleared lysateswas subjected to electrophoresis in 0.7% agarose(Seakem, Marine Colloids, Inc.) dissolved in Tris-borate buffer (89 mM Tris base, 2.5 mM disodiumEDTA, and 8.9 mM boric acid). A dye solution con-sisting of bromophenol blue (0.07%), SDS (7%), andglycerol (33%) in water was added at 5 Al per sampleto DNA samples prior to electrophoresis. Electro-phoresis was carried out in a vertical lucite slab gelapparatus (9). The dimensions of the gel were 9.6 by14.2 by 0.6 cm. Sample wells were made by use of aLucite comb with 14 teeth, each 0.508 cm wide andspaced by 0.478 cm. The power source was a Heath-kit regulated high-voltage power supply, model 1P-17, and electrophoresis was carried out at 60 mA, 120V, for 2 h or until the dye neared the bottom of thegel. The gel was then placed in a solution of ethid-ium bromide in water (0.4 ug/ml) and stained for 15min.

Electron microscopy of DNA. The contour lengthof plasmid DNA was determined as described previ-ously (7, 12). Molecular weights were calculatedfrom the contour lengths by using the conversionfactor of 1 ,Am = 2.07 x 106.

RESULTS AND DISCUSSIONRelationship of molecular weight to migra-

tion in agarose gels. Purified CCC plasmidDNAs from the previously characterized Rplasmids Rldrdl9, RP4, Sa, RSF1010 (Apll3),and RSF1030, as well as monomeric and di-meric CCC molecules of the ColEl derivativepMB8, were subjected to agarose gel electropho-resis. The migrations of these CCC plasmid

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CHARACTERIZATION OF PLASMID DNA 1531

A B C D E F G H

FIG. 1. Agarose gel electrophoresis of plasmid DNA of known molecular weight. The plasmids wereisolated from E. coli K-12 sublines, each subline containing one plasmid. The plasmid DNA was purified bycesium chloride-ethidium bromide centrifugation and fractionation, or was ethanol precipitated from acleared lysate prepared by SDS lysis and salt precipitation. Migration was from top (cathode) to bottom(anode). (A) DNA (25 ,u) purified by cesium chloride-ethidium bromide density centrifugation and fractiona-tion; top band, RSF1010 Ap113 (molecular weight, 8.7 x 106); middle band, dimer ofpMB8 (molecularweight, 3.74 x 106); bottom band, pMB8 (molecular weight, 1.8 x 106); from E. coli K-12 sublines growntogether in same culture flask. (B) Rldrdl9, 25 pl ofpurified DNA. (C) Sa (molecular weight, 23 x 106), 25 1dofpurified DNA. (D) Upper band, R1; middle band, RP4 (molecular weight; 34 x 10f), and lower band, Sa;10 pl ofpurified DNA of each plasmid species. (E) Ethanol-precipitated DNA (25 PI) of Sa; diffuse band ischromosomal fragments in this and following wells. (F) Ethanol-precipitated DNA (15 pi) of RSF1030(molecular weight, 5.5 x 106). (G) Ethanol-precipitated DNA (15 pi) ofRP4 (upper band) and Sa (lowerband) grown together in same culture flask. (H) Ethanol-precipitated DNA (30 pu) ofRP4.

DNA species were related inversely to theirmolecular weights (Fig. 1A through F); i.e., thelarger the molecular weight, the slower therate of migration. In each instance, only a sin-gle well-defined band of DNA was observed foreach plasmid species. That the DNA was re-tained in the CCC state was confirmed by cut-ting out the DNA band from the gel, eluting theDNA from the agarose, and examining theDNA in an electron microscope. Neither opencircular (OC) DNA, which migrates consider-ably slower than CCC DNA (1, 9), nor linearDNA, which generally migrates faster than

CCC DNA (1, 9), was observed as long as theDNA was freshly prepared or had been storedat -20°C prior to use. Repeated freezing andthawing of the DNA samples or mild deoxyribo-nuclease treatment could convert the DNA toboth the OC and linear form, however. Presum-ably, the presence of a "relaxation complex" (3)could result in detectable amounts of OC DNAmolecules, although this has not been a prob-lem in our experience thus far.A plot of the logarithm of relative migration

of the standard CCC plasmid DNA prepara-tions through the gel versus the logarithm of

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1532 MEYERS ET AL.

1062 3 4 5 6 7 891

Relative mobilil

FIG. 2. Molecular weight vers,ofplasmid DNA from E. coli K-iplasmids ofknown molecular weiplasmid Rl (molecular weight,arbitrarily at 10. Symbols: 0, Rl62 x 106); *, RP4 (molecular weSa (molecular weight, 23 x 10f);(molecular weight, 8.7 x 106); 0,

lar weight, 5.5 x 106); O, pMBTweight, 3.74 x 106); *, pMB81.87 x 106).

plasmid molecular weightmined by electron microscophysical measurements; 7) is Elinear curve was observed corthe difficulties inherent in tplasmid molecular weight fro]gration is shown in Fig. 1, weD contains a mixture of Rldrplasmid DNA and representsconfiguration that we attem]accurate measurement. Wellcess of Sa CCC DNA, and itthat this higher concentrationat a position noticeably lowerSa DNA (the lower band) in wrelative migration of the Sa Dstandard, therefore, the estinlecular weight of the plasmifrom Fig. 1 would be somewl

experience, however, the degdifferences in the amount (greater than 10% of the true p

weight (assuming contourments as the standard forweight).Plasmid DNA isolated by c

tion of DNA from an SDS-si(Fig. 1E through H) compared

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observed with DNA purified by cesium chlo-ride-ethidium bromide density gradient centrif-ugation. Chromosomal DNA was present inthese preparations but generally appeared as adiffuse band well below CCC plasmid DNA, 10x 106 daltons in mass. The chromosomal frag-ments also did not interfere with the detectionofCCC plasmid DNA of the 5.5 x 106 molecularweight plasmid RSF1030 (Fig. 1F) or plasmidsof lower molecular weight. Even in the pres-ence of very large amounts of contaminatingchromosomal fragments it was unusual forplasmid DNA to be obscured. Nevertheless, itshould be noted that this is a potential draw-back of the method examining such partiallypurified cell lysates, which contain plasmids of

20 3040 '60'801C 0 from 7 x 106 to 15 x 106 daltons in mass. Aty comparison of the plasmid DNA from clearedsrvm

lysates ofE. coli K-12 (Fig. 1E, G, and H) with2 sublines carrming the standard purified CCC DNAs (Fig. 1D)ght. The mobility of clearly demonstrates the utility and practical-62 x 106) was set ity of the method.(molecular weight, Application of the agarose gel electrophore-?ight, 34 x 106f); A, sis to clinical isolates and its use in geneticA, RSF1010 Api13 analysis. The agarose gel electrophoresisRSF1030 (molecu- method has been applied to the analysis of the9 dimer (molecular plasmid complement of clinical isolates. Three(molecular weight, toxigenic E. coli isolates from individuals par-

ticipating in a study of traveller's diarrhea inMexico were received from E. Gangarosa, and

previously deter- their plasmid complement was investigated bype analysis and the agarose gel method as well as the standard3hown in Fig. 2. A cesium chloride-ethidium bromide density gra-nsistently. One of dient centrifugation method. One strain, 514,the estimation of was characterized as producing a heat-stablem its relative mi- enterotoxin (ST) and was resistant to tetracy-,lls C and D. Well cline (Tc) and sulfonamide (Su). Strain 471 alsod19, RP4, and Sa produced ST, whereas strain 322 produced boththe optimal band ST and a heat-labile (LT) enterotoxin. Strainspt to achieve for 471 and 322 were susceptible to clinically rele-C contains an ex- vant antibiotics. Figure 3 shows the migrationmay be observed of DNA extracted from these clinical isolates.ofSa DNA bands Figure 3A shows the DNA from an E. coli K-12

r than that of the strain, showing only chromosomal fragments;rell D. Taking the Fig. 3B shows the migration of standard CCCNA in well D as a plasmid species. The latter sample was runnation of the mo- routinely in every gel as a basis for the calcula-i DNA in well C tion for the molecular weight of unknownhat lower. In our plasmid species. DNA from an E. coli K-12r,ee of error from F- strain or an E. coli K-12 Rldrdl9 strainof DNA was no was prepared each time to monitor the effec-)lasmid molecular tiveness of the lysis procedure and ethanol pre-length measure- cipitation. In addition, Fig. 3 shows that each oftrue molecular the clinical isolates contained at least two plas-

mid species and that the plasmid DNA mi-sthanol precipita- grated to the same position in the gel whetheralt-cleared lysate the plasmid DNA was purified by cesium chlo-favorably to that ride-ethidium bromide centrifugation or was

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CHARACTERIZATION OF PLASMID DNA 1533

A B CD EF G H IJ

FIG. 3. Agarose gel electrophoresis of plasmid DNA from enterotoxigenic E. coli. (A) Chromosomalfragments from E. coli K-12 F-; 15 pd ofethanol-precipitated DNA from cleared lysate. (B) Purified standardDNA (13 gil): upper band, R1drdl9; second band, RP4; third band, Sa; fourth band, RSF1010 Ap113; fifthband, dimer ofpMB8; sixth band, pMB8. (C) E. coli 514 ST+ Tcr Sur; 15 pi ofethanol-precipitated DNA fromcleared lysate. (D) E. coli 471, 20 p1 of DNA purified by cesium chloride-ethidium bromide densitycentrifugation and fractionation. (E) E. coli 322 ST+ LT+; 30 pl of ethanol-precipitated DNA from clearedlysate. (F) E. coli 514,30 p1 ofethanol-precipitated DNA from cleared lysate. (G) E. coli 471; 30 1d ofethanol-precipitated DNA from cleared lysate. (H) E. coli 322; 30 pi ofDNA purified by cesium chloride-ethidiumbromide density centrifugation and fractionation. (I) E. coli 514; 15 pl ofDNA purified by cesium chloride-ethidium bromide density centrifugation and fractionation. (J) E. coli 471; 30 pl ofDNA purified by cesiumchloride-ethidium bromide density centrifugation and fractionation.

simply ethanol precipitated from a cleared ly-sate (cf. Fig. 3C, F, and I; D, G, and J; 3E andH).The purified plasmid DNA samples from

strains 514, 471, and 322 were examined in anelectron microscope. Isolated OC moleculeswere photographed and their contour lengthswere measured (Table 1). The molecularmasses of the plasmids of these strains werealso estimated from their migration in gels rel-ative to the standard plasmid DNA. The largestmolecular species in the clinical isolates pos-sessed a mass greater than the largest standardplasmid species (Rldrdl9), 62 x 106 daltons.However, when the standard curve was pro-

jected as a straight line beyond the molecularmass of Rldrdl9, the relative migration of thelarger plasmid species of strains 514, 471, and322 fell precisely on the projected line (Fig. 4).Table 1 permits a comparison of the molecularmass obtained by electron microscopy and thatestimated from migration in gels. There was aclose agreement between the two methods.The observation that enterotoxigenic clinical

isolates possessed several distinct plasmid spe-cies was by no means surprising. Earlier stud-ies from this and other laboratories establishedthat this is a common occurrence (6, 18). In thefurther analysis of such strains, it is generallynecessary to mate the clinical isolate with a

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TABLE 1. Comparison of molecular weightdeterminations by agarose gel electrophoresis andcontour length measurements ofplasmids from

enterotoxigenic E. coli

Contour length Gel electrophoresis

EstimatedStrain No. of Mol Wta No. of de- mOl Wt

molecules termina- ( 01measured (x 1061) tions from rela-

tive mi-gration

514 27 91.7 ± 2.5 3 9077 61.4 ± 2.1 3 61.5-63

322 14 93.2 ± 3.0 3 94-9535 43.9 ± 1.9 3 44-47

471 13 67.5 ± 2.3 3 70-7215 44.6 ± 2.1 3 46-4717 3.2 ± 0.21 3 3.4-3.6

a Molecular weights were calculated from contourlengths by using the conversion factor 1 gm = 2.07x 106.

standard laboratory recipient and to screentransconjugants for the inheritance oftoxigene-sis, (Ent+) and other possible plasmid-mediatedproperties such as antibiotic resistance or Kantigen synthesis. We have found the agarosegel method to be a useful adjunct for suchscreening. For example, toxigenic strain B41,isolated from scouring calves (17, 18), encodesfor ST and confers resistance to streptomycin(Sm) and Tc. When strain B41 was analyzed byagarose gel electrophoresis it was observed tocontain two plasmid species estimated to be 65x 106 and 46 x 106 daltons (Fig. 5B). Genetictransfer experiments between strain B41 andE. coli K-12 strain 711 resulted in the isolationof Ent ST+ Tcs Sms transconjugants as well asEnt ST- Tcr Smr transconjugants. The analysisof the DNA from these transconjugants by aga-rose gel electrophoresis revealed that the EntST+ Tcs ss transconjugants harbored only the65 x 106-dalton plasmid (Fig. 5A), whereas theEnt ST- Tcr Smr transconjugants possessedonly the 45 x 106-dalton plasmid species (Fig.5C). Ent STr Tcr Smr transconjugants possessedboth plasmids (Fig. 5B). Hence, the applicationof the agarose gel method permitted not onlythe identification of the plasmid species withinstrain B41, but also served as an invaluable aidin quickly determining the association betweenthe plasmid species and a specific phenotypictrait. In a similar vein, the 61 x 106-daltonplasmid of the human toxigenic isolate 514(Fig. 3C, F, and I) has been associated withtransmissible Tc Su resistance, whereas thelarger plasmid species, 90 x 106 daltons, ap-pears to be solely associated with the Ent phe-notype. We applied this methodology as an ad-

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junct to the study of R plasmids of microbialspecies resident upon patients in a burn unit(L. P. Elwell and B. Minshew, unpublisheddata).The agarose gel method has been successfully

applied to clinical isolates ofE. coli, Klebsiellapneumoniae, Citrobacter freundii, Shigella dy-senteriae, Enterobacter cloacae, Salmonellasp., and other enterobacteria. As illustrated inFig. 6 and Table 2, we also found the method tobe directly applicable to strains of H. influ-enzae bearing R plasmids, as well as to clinicalisolates ofN. gonorrhoeae. The method may beapplicable to many bacterial species of interestand, indeed, in several isolated instances weused the method to detect plasmid DNA instrains of Bacteroides fragilis and Streptococ-cus faecalis.The agarose gel electrophoresis method for

detecting CCC plasmid DNA and the estima-tion of plasmid mass is sensitive, does not re-quire radioisotopes or ultracentrifugation, andcompares favorably with electron microscopy ofpurified plasmid DNA. The method is rela-tively simple in that it employs readily availa-ble reagents and does not require, by currentstandards, sophisticated equipment. Althoughwe employ a rather elaborate ultraviolet lighttransilluminator and camera system to recordour results, in point of fact our initial data wereobtained with a hand-held "blacklight" and amillimeter ruler. Undoubtedly, the basic meth-

Rellive mobility

FIG. 4. Molecular weight versus relative mobilityofstandard plasmid DNA from E. coli K-12 sublinesand from toxigenic E. coli clinical isolates. The mo-bility ofRldrdl9 (molecular weight, 62 x 106) wasset arbitrarily at 10. Symbols: 0, standard plasmidDNA as described in the legend of Fig. 2; 0, plas-mids from E. coli 322; M, plasmids from E. coli 514;A, plasmids from E. coli 471.

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CHARACTERIZATION OF PLASMID DNA 1535

CaB C

FIG. 5. Electrophoresis ofplasmid DNA from E. coli K-12 transconjugants derived from a mating withtoxigenic E. coli B41. (A) Ethanol-precipitated DNA (25 ,d) from cleared lysate of Tc' Sms ST+ transconju-gant. (B) Ethanol-precipitated DNA (25 p) from cleared lysate of Tcr Cmr ST+ transconjugant. (C) Ethanol-precipitated DNA (25 p1) from cleared lysate of Tcr Smr transconjugant.

odology can be substantially simplified and re-fined. For example, ribonuclease digestion canbe eliminated with no serious interference inDNA detection, and the time required forethanol precipitation can be shortened substan-tially where maximal DNA recovery is not acritical factor. The method is also of considera-ble utility since from 8 to 10 cultures can beeasily analyzed simultaneously by a single in-vestigator within 3 days.

Despite the relative simplicity of the method,there are several important limitations. Theeffect of DNA concentration on gel migrationhas already been noted, as has the potentialinterference with the detection of some classesof CCC plasmid species by contaminating chro-mosomal fragments. Moreover, the amount ofCCC plasmid DNA, as well as the concentra-tion of contaminating chromosomal fragments,may vary from sample to sample, particularly

with clinical isolates. For these reasons, weoften initially run several different volumes(commonly 5, 10, and 25 ,1u) of material ob-tained from the ethanol precipitation of clearedlysates. Although the concentration of DNA inthese samples is not known, as little as 50 ng ofDNA in the band of least visibility need bepresent for the band to be seen (1, 9). As notedearlier, we have not experienced any difficultythus far with OC DNA molecules as long as theDNA has been freshly prepared. We presumethat, in part, this is a reflection that OC mole-cules are normally present in relatively lowconcentrations in our preparations and migrateconsiderably more slowly than the correspond-ing CCC form (1, 9). Nevertheless, we empha-size that the gel method alone does not permitdiscrimination between OC and CCC DNA. Forexample, in a previously uncharacterized iso-late, one might not be able to discern between

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FIG. 6. Agarose gel electrophoresis of ethanol-precipitated plasmid DNA from cleared lysates of N.gonorrhoeae and H. influenzae. (A) N. gonorrhoeae (GSF1911): molecular weight, 2.65 x 106; crypticplasmid. (B) N. gonorrhoeae (GFF1990): molecular weight, 2.5 x 106; cryptic plasmid. (C) Standard plasmidDNA as described in the legend to Fig. 2. (D) Chromosomal DNA from E. coli K-12, lacking any plasmidDNA. (E) H. influenzae (UB2811); molecular weight, 31 x 106; plasmid specifying Tetr. (F) H. influenzae(RSF0885): molecular weight, 3.6 X 106; plasmid specifying Ampr. (G) H. influenzae (RSF007): molecularweight, 30 x 106; plasmid specifying Ampr. (H) H. influenzae 4B, chromosomal DNA only.

TABLE 2. Comparison of molecular weight determinations by agarose gel electrophoresis and contour lengthmeasurements ofplasmids from N. gonorrhoeae and H. influenzae

Contour length Gel electrophoresis

Strain and plasmid No. of mole- No. of deter- Estimated mol wtcules mea- Mol wta (x 106) minations (x 106) from relative

sured migration

N. gonorrhoeae (GSF1911) 109 2.39 ± 0.04a 2 2.4-2.65N. gonorrhoeae (GSF1990) 387 2.36 ± 0.12a 2 2.3-2.5H. influenzae (UB2811Pb 3 31.1 1 30.0H. influenzae (RSF0885) 56 3.19 ± 0.12' 1 3.6H. influenzae (RSF007) 56 30.2 ± 0.59( 1 30.0

a Data taken from reference 16.b Clinical isolate received from J. Saunders and M. H. Richmond.e Data taken from reference 5.

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CHARACTERIZATION OF PLASMID DNA 1537

two high-molecular-weight species of CCCDNA or one species that is easily nicked. Ifthere is doubt on this point, it is possible tosubject the lysate to freezing and thawing, milddeoxyribonuclease treatment (3), or X irradia-tion (8, 12) to deliberately convert the CCCDNA to the OC form. It should also be empha-sized that, although we have easily detectedplasmids ranging from 0.6 x 106 to 95 x 106 inmolecular weight, the SDS-salt precipitationmethod is considerably less effective for higher-molecular-weight plasmids (10). Also, plasmids120 x 106 daltons in mass or higher do notpenetrate the gel under the stated experimen-tal conditions. The latter drawback can be over-come by employing horizontal electrophoresiswith gels containing a concentration of 0.35%agarose (D. Merlo and J. Meyers, unpublisheddata). Our choice of the SDS-salt precipitationmethod was dictated by our familiarity withthe technique, as well as its utility in success-fully lysing a wide variety of microbial species(10). However, the PEG precipitation method(15), as well as the Brij 58- or Triton X-100-cleared-lysate method (3), could be readily sub-stituted.The agarose gel electrophoresis method is not

presented here as a means for precise plasmidcharacterization. It will, however, often rapidlyand simply provide all ofthe information that isrequired by an investigator with regard to theplasmid complement of a bacterial strain. Wefound the method to be an important adjunct togenetic analysis, and it is invaluable for surveywork as well as the epidemiological investiga-tion of plasmid dissemination.

ACKNOWLEDGMENTSThis work was supported by contract FDA-223-73-7210

from the Food and Drug Administration. L. Elwell is sup-ported by National Research Service Award GM-07187 fromthe National Institute of General Medical Sciences.We wish to thank H. W. Boyer and M. Betlach for their

good advice. We would also like to thank Linda Ehnes,Julie Sasewich, and Martha Webb for their excellent tech-nical assistance.

LITERATURE CITED1. Aaij, C., and P. Borst. 1972. The gel electrophoresis of

DNA. Biochim. Biophys. Acta 269:197-200.

2. Boyer, H. W. 1974. Restriction and modification ofDNA: enzyme substrates. Fed. Proc. 33:1125-1127.

3. Clewell, D. B., and D. R. Helinski. 1969. Supercoiledcircular DNA-protein complexes in Escherichia coli:purification and induced conversion to open circularDNA form. Proc. Natl. Acad. Sci. U.S.A. 62:1159-1166.

4. Crosa, J., L. K. Luttropp, and S. Falkow. 1975. Natureof R-factor replication in the presence of chloram-phenicol. Proc. Natl. Acad. Sci. U.S.A. 72:654-658.

5. Elwell, L. P., J. de Graaff, D. Seibert, and S. Falkow.1975. Plasmid-linked ampicillin resistance in Haemo-philus influenzae type b. Infect. Immun. 12:404-410.

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