The covalently closed form of circular duplex SV40 DNA was separated from the open and linear form of SV40 DNA by agarose gel electrophoresis in a large-scale gel system. The closed circular DNA was recovered from agarose gels by re-electrophoresing the gel slices. The recovery of DNA was about 70%. Electron microscopic analysis showed that the recovered DNA did not have doube- or single-stranded breaks. The recovered DNA can be used without further purification for electron microscopy, as a substrate for experiments using restriction endonuclease and as a template for in vitro RNA synthesis.</p

Hidaka, Hideyuki

Oda, Takuzo

Tanabe, Naoko

Watanabe, Sekiko

Okayama University Digital Information Repository

A simple large-scale method for separating closed circular form DNA by gel electrophoresis

Okayama University Scientific Achievement Repository

Acta Medica OkayamaVolume 32, Issue 6 1978 Article 1DECEMBER 1978A simple large-scale method for separatingclosed circular form DNA by gelelectrophoresisNaoko Tanabe∗ Hideyuki Hidaka†Sekiko Watanabe‡ Takuzo Oda∗∗∗Okayama University,†Okayama University,‡Okayama University,∗∗Okayama University,Copyright c©1999 OKAYAMA UNIVERSITY MEDICAL SCHOOL. All rights reserved.A simple large-scale method for separatingclosed circular form DNA by gelelectrophoresis∗Naoko Tanabe, Hideyuki Hidaka, Sekiko Watanabe, and Takuzo OdaAbstractThe covalently closed form of circular duplex SV40 DNA was separated from the open andlinear form of SV40 DNA by agarose gel electrophoresis in a large-scale gel system. The closedcircular DNA was recovered from agarose gels by re-electrophoresing the gel slices. The recoveryof DNA was about 70%. Electron microscopic analysis showed that the recovered DNA did nothave doube- or single-stranded breaks. The recovered DNA can be used without further purifica-tion for electron microscopy, as a substrate for experiments using restriction endonuclease and asa template for in vitro RNA synthesis.KEYWORDS: separating method, simian virus 4D DNA, closed circular DNA, gel electrophore-sis∗Copyright c©OKAYAMA UNIVERSITY MEDICAL SCHOOLPMID: 218425 [PubMed - indexed for MEDLINE]Acta Merl. Okayama 32, (6), 379-385 (1978)A SIMPLE LARGE-SCAL:E~METHOD FOR SEPARATINGCLOSED CIRCULAR FORM DNA BY GELELECTROPHORESISNaoko TANABE, Hideyuki HIDAKA, Sekiko WATANABEand Takuzo aDADepartment of Biochemistry, Cancer Institute, Okayama UniversityMedical School, Okayama 700, JapanReceived July 21, 1978Abstract. The covalently closed form of circular duplex SV4DDNA was separated from the open and linear form of SV4D DNA byagarose gel electrophoresis in a large-scale gel system. The closedcircular DNA was recovered from agarose gels by re-electrophoresingthe gel slices. The recovery of DNA was about 70%. Electron micro-scopic analysis showed that the recovered DNA did not have double- orsingle-stranded breaks. The recovered DNA can be used without fur-ther purification for electron microscopy, as a substrate for experimentsusing restriction endonuclease and as a template for in vitro RNA syn-thesis.Key words: separating method, simian virus 4D DNA, closedcircular DNA, gel electrophoresisAgarose gel electrophoresis is a versatile technique for rapid resolution ofvarious size and form DNAs (1-5). It is extremely important for studies of thestructure and function of simian virus 40 (SV40) DNA to obtain the closed circu-lar form of SV40 DNA (6-8). Closed circular DNA is usually purified by longperiod of ultracentrifugation in CsCl ethidium bromide equilibrium gradients,and ethidium bromide intercalated into DNA must be excluded completely bysome procedure (6-8). We applied agarose gel electrophoresis to separation ofthe closed form of circular duplex SV40 DNA from the open and linear forms ofSV40 DNA in a large-scale gel system. In this paper we show that the presentmethod is a simple and satisfactory method for obtaining closed circular DNA.MATERIALS AND METHODSDNA preparation. CV-l cells (a cell line established from African greenmonkey kidney cells) were infected with SV40 at a multiplicity of 0.1 to 0.5plaque forming units per cell and harvested 7 to 9 days after infection. SV40 waspurified by the method of Yoshiike (9). SV4D DNA was isolated from the puri-fied virions by the method of Marmur (10). The SV4D DNA was separated intothree components: closed circular form (SV4D DNA I), open circular form (SV4D3791Tanabe et al.: A simple large-scale method for separating closed circular formProduced by The Berkeley Electronic Press, 1978380 N. TANABE et at.DNA II) containing linear form DNA (SV40 DNA III) and defective DNA byutilizing difference in the electrophoretic mobility in agarose gel as described inthe next section.Separation of closed circular DNA. Electrophoretic separation was performedin 0.7% (wjv) agarose gels set in two cylindrical glass tubes of 0.6 cm and 2.7 cminternal diameter (25 cm lengths). Agarose gel set in a 0.6 by 25 cm tube wasused for monitoring migration of SV40 DNAs, the other gel in a 2.7 by 25 cmtube for separation of SV40 DNAs. The lower ends of the gel tubes werecovered with a dialysis membrane. Agarose (Nakarai Co.) was dissolved in elec-trophoresis buffer (0.09 M Tris, 2.5 mM NazEDTA, 20 mM sodium acetate, 18 mMNaCl, pH adjusted to 8.1 at 25°C with glacial acetic acid), refluxed for 30 min at100°C, then cooled to 45°C and poured into glass tubes in an incubation roomcontrolled at 37°C. The gels were allowed to stand for about 12 h and the upperend of the gels were extruded and sliced evenly prior to use.DNA samples for electrophoresis were mixed with 15% (wjv) sucrose (DNase-free) and 0.01 % (wIv) bromophenol blue added as tracking dye. Usually, 25 pIand 500 pI of samples (200 pg DNA/ml) were applied to monitoring and separatinggels, respectively, but 800 pI sample containing up to 100 pg of DNA could beused for separating gel without any significant reduction in resolution.The sample were run for 24 to 30 h at 4°C and 1.5 V /cm in a vertical tankwith the gels fully immersed in buffer. In order to visualize DNA in the moni-toring gel, the gel was extruded into a solution containing 1pg of ethidium bromideper ml of electrophoresis buffer. After 30 min the stained bands were visualizedby long wavelength ultraviolet light (about 365 nm). The gel containing theclosed circular DNA was excised, using the visualized monitoring gel as amarker. The gel was cut into small pieces and placed in the conical portion ofa 20 ml glass pipette that had been cut off to make a volume of about 20ml. Thetip of the pipette was plugged with glass wool. A dialysis bag was slipped overthe tip of the pipette and the DNA was electrophoresed out of the gel into dialy-sis bag at 2.0 mA/tube for 24 h at 4°C using electrophoresis buffer. At the end ofelectrophoresis the polarity of the current was reversed for 2 min to preventtrapping of the DNA in pores of the dialysis membrane. The solution containingDNA was recovered from the dialysis bag and dialyzed against appropriatebuffer.Molecular weight determination of separated SV40 DNA I. Four fragments oflambda DNA were used as a standard in estimating the molecular weight ofseparated DNA. These fragments were prepared from six fragments of phagelambda DNA by Eco RI restriction endonuclease. The molecular weights werel3.7x (A-), 4.6x (B-), 3.0x (E-) and 2.1 X 106 (F-fragment) (5). The separated SV40DNA I (closed circular form) was previously cleaved into the linear form by EcoRI endonuclease (11) and subjected to electrophoresis in 0.5% (w/v) agarose-1.7%(wIv) polyacrylamide composite gel prepared by the method of Peacock andDingman (12). The samples were run for 4 h at 20°C and 4 V /cm in Tris-boratebuffer (12).Electron microscopy. Separated SV40 DNAs (bands I, II-III and IV) werespread by the Kleinschmidt protein monolayer technique (13). The spreading2Acta Medica Okayama, Vol. 32 [1978], Iss. 6, Art. 1http://escholarship.lib.okayama-u.ac.jp/amo/vol32/iss6/1Electrophoretic Separation of Closed Circular DNA 381solution contained 1 M ammonium acetate, 2 mM NazEDTA, pH 7.5, 1% form-aldehyde and 0.01 % cytochrome C, and the subphase contained 250 mM ammo-nium acetate and 2 mM NazEDTA (pH 7.5). The protein-nucleic acid film waspicked up on carbon-coated copper grids and dried in ethanol and isopentane.The grids were rotary shadowed with Pt/Pd alloy (w/W, 8/2) and examined in aHitachi electron microscope (HU-ll D).RNA synthesis with separated SV40 DNA I as template. RNA synthesis wascarried out for 30 min at 3rC in a reaction medium of 200 (11 containing 180 mMKCl, 33 mM Tris-HCl (pH 7.9), 6 mM 2·mercaptoethanol, 3.3 mM MgClz, 0.165mM ATP, CTP, GTP and [ 3zPJUTP, 2/tg SV40 DNA I and 3/1g or 5 fig RNApolymerase (Boehringer Manheim). The substrate [32PJUTP was~abeledwith3Zp in the alpha position (specific activity: 50 to 125 Ci/mmol; RadiochemicalCenter, Amersham). Synthesized RNA products were analyzed by a slab gel inthe Tris/EDTA/borate system of Peacock and Dingman (12). The electropho-resed slab gel was exposed to Sakura medical X-ray film for 24 h at 4°C.RESULTSFig. 1 shows a representative pattern of SV40 DNA separated by separating(Fig. 1a) and monitoring gels (Fig. 1b). It was confirmed by the procedures de-scribed below that the closed form of circular SV40 DNA (SV40 form-I DNA)was contained in the band I of Fig. L After electrophoresis, the DNA containedin the band I was recovered using the procedure described in Materials and Me-thods.The DNA recovered from band I was analyzed for molecular weight by gelelectrophoresis and was checked for closed circular form molecule under theelectron microscope. The DNA recovered from band I in Fig. la was sized onthe basis of its electrophoretic mobility compared to the mobilities of four lambdaDNA fragments of known molecular weights (Fig. 2) and was about 3Ax 106, asshown in Figs. 3. Figs. 4a and 4b show representative electron micrographs ofSV40 DNAs recovered from the bands I and II-III, respectively, in Fig. la.Although there are some open circular form and linear form DNAs in Fig. 4a,the molecular forms were interpreted as arising during preparation of samplesfor electron microscopy rather than contaminating in the gels, because the opencircular form and linear form DNA differ in electrophoretic mobility from theclosed circular form DNA, as represented in Figs. 1, 4a, and 4b, and in the studiesof Dingman et at. (14). Therefore, DNA contained in band I is a complete andcovalently closed circular molecule. Fig. 4c shows a representative electronmicrograph of SV40 DNA recovered from band IV in Fig. la. It was confirmedby electron microscopy and gel electrophoresis that the DNA molecule was de-fective in molecular weight.To check whether the recovered DNA can be used without further purifica-tion we performed two types of experiments. One was electron microscopy of3Tanabe et al.: A simple large-scale method for separating closed circular formProduced by The Berkeley Electronic Press, 1978382aFig. 1,bN. TANABE et at.II-IIIIVFig. 2.ABSV40EFFig. 1. Electrophoretic patterns of SV40 DNA molecules (SV40 DNA I, II-III and IV)separated in 0.7% separating agarose gel (a) and 0.7% monitoring agarose gel (b). Gels a and bwere set in two cylindrical glass tubes of 2.7 cm and 0.6 cm internal diameter (25 cm length),respectively. Migrations were from top (cathode) to bottom (anode). Band I, closed circularDNA (SV40 DNA I); II-III, open circular DNA (SV40 DNA II) slightly containing linear DNA(SV40 DNA III); and IV, closed circular form of defective SV40 DNA (defective SV40 DNA).Fig. 2. Estimation of molecular weight of SV40 DNA recovered from band I representedin Fig. 1 by gel electrophoresis. Recovered SV40 DNA (closed circular molecule) was cleavedby Eca RI endonuclease according to the procedure described in the text prior to gel analysisand was converted into linear DNA. SV40 DNA migrated from top (cathode) to bottom(anode) with four lambda DNA fragments: A, 13.7x; B, 4.6x; E, 3.0x; and F, 2.1 X 106•Gel was composed of 0.5% agarose and 1.7% acrylamide.4Acta Medica Okayama, Vol. 32 [1978], Iss. 6, Art. 1http://escholarship.lib.okayama-u.ac.jp/amo/vol32/iss6/1Electrophoretic Separation of Closed Circular DNAF, .;I- 107 \~2="'"~ B .J0:>': E '.\F •1060 0.5 1.0 1.5 2.0 2.5RELATIVE MOBILITY383Fig. 3. Relative electrophoretic mobilities of SV40 DNA (0, pointed with the arrow)and lambda DNA fragm~nts te, labeled with A, B, E and F) in agarose-acrylamide gel re-presented in Fig. 2. All points represent the relative mobility to the mobility of the largestlambda DNA fragment A produced by Eeo RI restriction endonuclease (the mobility of frag-ment A was arbitrarily set at 1.0). Linear form of SV40 DNA was obtained by digestion ofclosed circular SV40 DNA with Eeo RI endonuclese. The points represented as closed cir-cules were obtained by plotting the molecular weight of each DNA fragment produced byEeo RI endonuclease against the relative mobility of each fragment. Molecular weight ofSV40 DNA was estimated from the straight line by plotting the relative mobility of SV40DNA III.Fig. 4. Electron micrographs of SV40 DNA molecules recovered from gel bands I, II-IIIand IV represented in Fig. lao a, SV40 DNA from band I (closed circular form SV40 DNA);b, SV40 DNA from band II-III (open circular form SV40 DNA slightly containing linear formSV40 DNA); and c, SV40 DNA from band IV (closed circular form of defective SV40 DNA).><23,000,5Tanabe et al.: A simple large-scale method for separating closed circular formProduced by The Berkeley Electronic Press, 1978384a b-28s-18s- 5sN. TANABE et at.the DNA as described above. The other was an ex-amination of template activity for transcription. Fig.5 shows gel autoradiograms of in vitro synthesizedRNA products by E. coli RNA polymerase usingclosed circular SV40 DNA recovered from gel band Ias template. The autoradiograms indicate that SV40DNA recovered from gel posseses the same level inthe activity of RNA synthesis as that recovered fromCsCl-density gradient (7).The recovery of DNA from agarose gel waschecked by using purified SV40 DNA (SV40 DNA I)and lambda DNA (linear form DNA, III; molecularFig. 5. Autoradiograph of an agarose-acrylamide slab gelcontaining 32P-labeled RNA products transcribed from SV40DNA I in the in vitrJ RNA synthesis system. SV40 DNA I usedwas recovered from the gel band I represented in Fig. la bythe procedure described in the text. (al, a molar ratio ofSV40 DNA to RNA polymerase enzyme was 1/9; and (b), 1/5.weight 3) X 106 (5)), and estimated as approximately 70% to 75% on the basisof recovered DNA concentration by optical density at 260 nm.DISCUSSIONGel electrophoresis was used for separation of closed circular form SV40DNA from open circular and linear DNA molecules having the same molecularweight. Samples (800 fll) containing up to 100 pg of DNA could be used on oneseparating gel without any significant reduction in separation.Closed circular form SV40 DNA has been generally separated in CsCl-ethidium bromide equlibrium gradients by long period of high speed centrifuga-tion (6-8) and after separation it is necessary to remove compleatly ethidiumbromide from recovered DNA, because ethidium bromide inhibits enzymaticreaction, such as restiction endonuclease reaction or RNA synthesis reaction (6-8).The present method for separating closed circular form DNA requires noultracentrifugation and gives good yields. The separated DNA can be usedwithout further purification for electron microscopy, restriction enzyme degra-dation and in vitro RNA synthesis, which is especially useful for studies of thestructure and function of SV40 DNA (15). The present method can be used forseparation of various closed circular DNAs by modification of the gel concentra-tion and/or composition.6Acta Medica Okayama, Vol. 32 [1978], Iss. 6, Art. 1http://escholarship.lib.okayama-u.ac.jp/amo/vol32/iss6/1Electrophoretic Separation of Closed Circular DNA 385Acknowledg.llents. We thank Professor Haruo Ozeki, Kyoto University, for bacteriophagelambda used in this study and Ms. Tamie Yasui for generous supplies of cultured CV-l cellsand SV40. This work was supported by a Grant-in-Aid for Scientific Research from theMinistry of Education, Science and Culture of Japan.REFERENCES1. Thorne, H. V.: Electrophoretic characterization and fractionation of polyoma virus DNA.j. Mol. BioI. 24, 203-211, 1967.2. Takahashi, M., Ogino, T. and Baba, K.: Estimation of relative molecular length of DNAby electrophoresis in agarose gel. Biochim. Biophys. Acta 174, 183-187, 1969.3. Fisher, M. P. and Dingman, C. W.: Role of molecular conformation in determining theelectrophoretic properties of polynucleotides in agarose-acrylamide composite gels. Bio-chemistry 10, 1895-1899, 1971.4. Aaij, C. and Borst, P.: The gel electrophoresis of DNA. Biochim. Biophys. Acta 269, 192-200, 1972.5. Helling, R. B., Goodman, H. M. and Boyer, H. W.: Analysis of endonuclease R. Eco RIfragments of DNA from lambda bacteriophages and other viruses by agarose-gel electro-phoresis. j. Virology 14, 1235-1244, 1974.6. Sebring, E. D., Garon, C. F. and Salzman, N. P.: Superhelix density of replicating simianvirus 40 DNA molecules. j. Mol. BioI. 90, 371-379, 1974.7. Laub, O. and Aloni, Y.: Transcription of simian virus 40. V. Regulation of simian virus40 gene expression. j. Virology 16, 1171-1183, 1975.8. Lebowitz, P., Stern, R., Ghosh, P. K. and Weissman, S. M.: Specificity of initiation oftranscription of simian virus 40 DNA I by Escheri,hia coli RNA polymerase: Identificationand localization of five sites for initiation with [r-32P]ATP. j. Virology 22, 430-445, 1977.9. Yoshiike, K.: Studies on DNA from low-density particles of SV40. 1. Heterogeneousdefective virions produced by successive undiluted passages. Virology 34:, 391-401, 1968.10. Marmur, J.: A procedure for the isolation of deoxyribonucleic acid from microorganisms.j. MDI. BioI. 3, 203-218, 1961.11. Morrow, J. F. and Berg, P.: Cleavage of simian virus 40 DNA at a unique site by abacterial restriction enzyme. Proc. Natl. Acad. Sci. USA 69, 3365-3369, 1972.12. Peacock, A. C. and Dingman, C. W.: Molecular weight estimation and separation ofribonucleic acid by electrophoresis in agarose-acrylamide composite gels. Biochemistry 7,668-678, 1968.13. Kleinschmidt, A. K.: Monolayer techniques in electron microscopy of nucleic acid mole-cules. Methods Enzymol. 12, Part B. eds. S. P. Colowick and N. O. Kaplan, AcademicPress, New York. pp. 361-377, 1968.14. Dingman, C. W., Fisher, M. P. and Kakefuda, T.: Role of molecular conformation indetermining the electrophoretic properties of polynucleotides in agarose-acrylamidegels. II. Biochemistry 11, 1242-1250, 1972.15. Hidaka, H., Ocho, M., Tanabe, N. and Oda, T.: Regulation of transcription of SV40 DNAby chromosomal proteins of host cells. Proceeding 1977 Mol. BioI. Meet. japan, KyoritsuShuppan Co., Ltd., Tokyo. pp. 62-64, 1977.7Tanabe et al.: A simple large-scale method for separating closed circular formProduced by The Berkeley Electronic Press, 1978

A simple large-scale method for separating closed circular form DNA by gel electrophoresis

Abstract

Similar works

Full text

Available Versions

Okayama University Digital Information Repository

Okayama University Scientific Achievement Repository