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A rapid alkaline extraction procedure for screening recombinant plasmid DNA

A rapid alkaline extraction procedure for screening recombinant plasmid DNA Downloaded from https://academic.oup.com/nar/article-abstract/7/6/1513/2380972/ by Ed 'DeepDyve' Gillespie user on 05 October 2019 Volume 7 Number 61979 Nucleic Acid s Research H.C.Bimboim+ and J.Doly Laboratoire de Ge'ne'tique Moleculaire, Institut de Recherche en Biologie Moleculaire, F-75221 Paris Cedex 05, France Received 3 August 1979 ABSTRACT A procedure for extracting plasmid DNA from bacterial cells 1s described. The method 1s simple enough to permit the analysis by gel electrophoresis of 100 or more clones per day yet yields plasmid DNA which is pure enough to be digestible by restriction enzymes. The principle of the method is selective alkaline denaturation of high molecular weight chromosomal DNA while covalently closed circular DNA remains double-stranded. Adequate pH control is accomplished without using a pH meter. Upon neutralization, chromosomal DNA renatures to form an insoluble clot, leaving plasmid DNA in the supernatant. Large and small plasmid DNAs have been extracted by this method. INTRODUCTION Bacterial plasmid DNAs are widely used as cloning vehicles in re- combinant DNA research. After new plasmids are constructed, they may be isolated and characterized with respect to their size and restriction enzyme pattern by gel electrophoresis. One method for preparing plasmid DNA In a highly purified form involves gently lysis of bacterial cells, centrifugation to remove the bulk of the chromosomal DNA, and then banding of residual DNA in a cesium chloride gradient in the presence of ethidium bomide. Covalently closed circular (CCC) DNA binds a different amount of the dye than does open circular (OC) or linear DNA and is readily separated from the latter two forms of DNA (1). A second, more rapid, method for preparing plasmid DNA employs hydroxyapatite chromatography (2); this also produces highly purified DNA. However, for many purposes including analysis by gel electrophoresis, less purified DNA can be used. Under favourable conditions, a colony of cells can be lysed and plasmid DNA can be detected in crude extracts after electro- phoresis (3-6); this permits the analysis of many clones and makes screening possible. In this report we describe another method for plasmid DNA extraction which is both simple enough to permit screening of many small samples, yet O Information Retrieval Limited 1 Falconberg Court London W1V5F G England 1613 Downloaded from https://academic.oup.com/nar/article-abstract/7/6/1513/2380972/ by Ed 'DeepDyve' Gillespie user on 05 October 2019 Nucleic Acids Research yield s plasnrid DNA in a form sufficientl y pure to be digestible by restriction enzymes or to be used to transform other cells. In these respects, the pro- cedure we describe is more versatile than other rapid extraction methods. Plasmid DNA can be detected in as littl e as 0.1 ml of non-amplified liquid cultur e or 1n a colony of cells scraped from a plate. PRINCIPLE OF THE ALKALINE EXTRACTION METHOD Previous workers have shown that there is a narrow range of pH (about 12.0-12.5) within which denaturation of linear DNA but not CCC-DNA occurs and that this property can be used for purifying CCC-DNA (7-10,20,21). We have utilize d this approach for developing a rapid extraction method for plasmid DNAs. Plasmid-containing cells are treated with lysozyme to weaken the cell wall and then lysed completely with sodium dodecyl sulfate (SDS) and NaOH. By choosing the ratio of cell suspension to NaOH solutio n carefully, a reproducible alkaline pH value 1s obtained without the necessity of monitoring the pH wit h a meter; further pH control is obtained by including glucose as a pH buffer. Chromosomal DNA, stil l in a very high molecular weight form, 1s selectivel y denatured ?nd when the lysate is neutralized by acidic sodium acetate, the mass of chromosomal DNA renatures and aggregates to form an insoluble network. Simultaneously, the high concentration of sodium acetate causes precipitation of protein-SDS complexes (11,12) and of high molecular weight RNA (13). In this way, most of the three major contaminating macro- molecules are co-precipitated and may be removed by a single centrifugation i n a bench-top centrifuge. Plasmid DNA (and residual low molecular weight RNA) are recovered from the supernatant by ethanol precipitation. Plasmid DNA may be analyzed by gel electrophoresis either intact in the CCC form or after digestion with a restrictio n enzyme. MATERIALS AND METHODS Cell strains and Media : Escherichia coli strains HB101 (14), SK1592 (15) and RRI (16) were used for most of the work reported here. E.coli F' strains GM218 (thr" , leu", arg", 1lv", thy", ura", his" , recA"/F'h1s+) and JM856 (thyA", lysA", pheA", arg", relA", recA'/F1 thy , lys , phe , arg , relA , tif ) from J . George were grown in minimal medium supplemented with the appro- priat e nutritional requirements at 30°C. Otherwise, L-broth was used through- out . When appropriate, Ampicillin (Ap) was added at a concentration of 100 ug/ml for liquid medium or 40 ug/ml for plates. Tetracycline (Tc) was used 1514 Downloaded from https://academic.oup.com/nar/article-abstract/7/6/1513/2380972/ by Ed 'DeepDyve' Gillespie user on 05 October 2019 Nucleic Acids Research fo r agar plates at a concentration of 10 ug/ml. Equipment and Reagents for Plasmid Extraction : Eppendorf-type (1.5 ml) polypropylene tubes and a bench-top centrifuge capable of generating 8-10,000 x g were used. A rack holding 60 Eppendorf tubes speeds up handling of larger numbers of samples. Several racks can be prepared by stacking 5 sheets of 2 nrn-thick aluminum (or other metal) and drillin g 11 mm-d1a. holes at 15 mm spacing through all simultaneously; the edges of each sheet are folded as legs. For complete removal of supernatants following centrifugation in Eppendorf tubes, a Pasteur pipette drawn out to a fine tip is used. Reagents : I. Lysozyme solution - 2 mg/ml lysozyme, 50 mM glucose, 10 mM CDTA, 25 mM Tris-HC l (pH 8.0). Prepare fresh daily from crystalline lysozyme and stock solutions of the other components. Store at 0°C. II . Alka- lin e SDS solution - 0.2 N NaOH, IS sodium dodecyl sulfate (SDS). Store at room temperature; stable for about 1 week. III. High salt solution - 3 M sodium acetate (pH 4.8). Prepare by dissolving 3 moles of sodium acetate in a minimal volume of water, adjusting to pH 4.8 with glacial acetic acid, and then adjusting volume to 1 1. Store at room temperature. CDTA (cyclohexane diamine tetracetate) 1s a chelating agent which is more soluble in alcohol and forms stronger complexes with metal ions than does EDTA. EDTA can be substituted fo r applications listed here. CDTA from Sigma or Aldrich Chemical Company was used. A stock solution of RNAse A (1 mg/ml in 5 mM Tris-HCl , pH 8.0) is treated by heating at 100° for 10 m1n. Standard Procedure for Extraction of Plasmid DNA from Small Volumes of Cell Cultures : (Footnotes refer to comments below.) The method has been used principally with plasmid pBR322 (ref. 16) and it s derivatives in E.coli strains HB101, RRI and SK 15921. Selected clones (ApR, TcS in the case of hybrid plasmids with inserts at the H1nd II I site of pBR322) are grown in 2.5 ml of L-broth containing 100 ug/ml Ap in 6-ml vials . After 18 h incubation, 0.5 ml of culture is transferred to a 1.5 ml Eppendorf tube for plasmid extraction , and the remainder is stored at -20°C after the addition of glycerol to 40J. All manipulations are carried out at room temperature unless otherwise Indicated. The tube is centrifuged for 15 seconds . The super- natant is carefully removed with a fine-ti p aspirator and the cell pellet is thoroughly suspended 1n 100 ul of solution I. After a 30 min. period of incubation at 0°C, 200 ml of solution I I 1s added and the tube is gently vortexed. The suspension should become almost clear and slightl y viscous. The tube is maintained for 5 min. at 0°C and then 150 ul of solution II I is 1515 Downloaded from https://academic.oup.com/nar/article-abstract/7/6/1513/2380972/ by Ed 'DeepDyve' Gillespie user on 05 October 2019 Nucleic Acids Research added. The contents of the tube are gently mixed by inversion for a few seconds during which time a clot of DNA forms. The tube is maintained at 0°C fo r 60 min. to allow most of the protein, high molecular weight RNA and chro- mosomal DNA to precipitate. Centrifugation for 5 m1n. yields an almost clear supernatant. Four-tenths of a ml of the supernatant is removed and trans- ferred to a second centrifuge tube . Small amounts of floating material may be carried over at this time. One ml of cold ethanol is added and the tube i s held at -20°C for 30 min. The precipitate 1s collected by centrifugation fo r 2 m1n. and the supernatant removed by aspiration. The pellet 1s dissolved 1n 100 ul of 0.1 M sodium acetate/0.05 M Tris-HCl (pH 8) and repredpitated with 2 volumes of cold ethanol. After 10 min. at -20°C, the precipitate is again collected by centrifugation as before. The pellet 1s dissolved in 40 ul water and then 10 ul of 5 x sample buffer 1s added. 10-20 ul is applied to an agarose gel for electrophoretic analysis. Modifications required when plasmid DNA is to be used for bacterial transformation or is to be treated by restriction enzymes : The pellet at the las t step is redissolved once more 1n 100 ul of 0.1 M sodium acetate/0.05 M Tris-HCl (pH 8) Instead of water and precipitated with 2 volumes of ethanol as before. For transformation, the pellet may be dissolved in 40 ul of water or dilute buffer and a few microliters added to competent cells. For restric- tio n enzyme analysis, the pellet 1s dissolved in 36 ul hLO and 4 ul of pan- creatic RNase (1 mg/ml) 1s added. After 30 min. incubation at 37°C, concen- trate d restriction enzyme buffer and 1 unit of enzyme are added. Following a period of digestion (37°, 60 min.), the sample is mixed with concentrated sample buffer and part of i t applied to a gel for electrophoretic analysis. Comments and other modifications of the method : 1. CCC-DNA in other cell types has been successfully extracted by the above method. Incubation at room temperature or 37°C with solution I may be necessary to disrupt Bacillus subtilis (S.D. Ehrlich, G. Rapoport - personal communication). A band corresponding in size to the 2 u circl e of yeast cells was detected by gel electrophoresis when alkaline extracts of Saccharomyces cerevisiae strain GRF18 protoplasts (kindly prepared by A. Hinnen) were examined. 2. One-half ml cultures have been grown directly in 1.5 ml Eppendorf tube ; alternatively, colonies (3-4 mm dia. ) can be scraped from the surface of an agar plate and resuspended in 100 ul of solution I. 3. If the cell number is very low, a longer centrifugation time may be required for the pellet to stick adequately to the wall of the tube or 0.2 ml of carrier cells can be added. 4. If the 1516 Downloaded from https://academic.oup.com/nar/article-abstract/7/6/1513/2380972/ by Ed 'DeepDyve' Gillespie user on 05 October 2019 Nucleic Acids Research cel l number is low, 25 ug of tRNA can be added as carrier to assist precipi- tatio n of plasmid DNA. Gel electrophoresis : 0.81 agarose gels were used, either as 6-tnn d1a. tubes or as 3-mm thick vertical slabs. Electrophoresis buffer contained 40 mM Tris , 20 mM sodium acetate, 2 mM EDTA, adjusted to pH 7.8 with acetic acid . 5 x sample buffer contained 25% sucrose, 5 mM sodium acetate, 0.05% bromophenol blue, 0.1% SDS. After electrophoresis, gels were stained with ethidium bromide (1 ug/ml) and photographed under UV illumination . Restrictio n Enzymes : Most were obtained from Biolabs and used under conditions described by the manufacturer. Hind II I was prepared at this institut e by A. Meier. Al l experiments Involving recombinant DNA were carried out with the permission of the French Commission on Recombinant DNA according to the French Guidelines. RESULTS AND DISCUSSION Treatment of plasmid DNA wit h alkali : The extraction method we have described involves exposing a crude extract to alkaline pH to denature chro- mosomal DNA. However, when covalently closed circular DNA is exposed to > pH 13, i t gives rise to a denatured form which is not readily renaturable (8,9) . The generation of this "irreversibly denatured" form (which is to be avoided in preparing plasmid DNA) is illustrated in Fig. 1. Fig. la shows the electrophoretic mobility of pBR322 DNA, purified by conventional methods (1). Band 4 is the supercoiled (CCC form). A small amount of 0C form (band 3) and CCC-dimer (band 2) are also visible. When treated with 0.1 N NaOH, neutralized, and run on a neutral gel, a faster moving band (band 5), corresponding to the "irreversibl y denatured" form, can be seen (Fig. lb). Two additional minor bands which migrate close to bands 4 and 5 are also seen. It is likely that these represent two single-stranded forms of plasmid DNA (linear and circular) which arise by denaturation of the 0C form. This was verified (Fig. lc and Id) by treating the CCC form with nuclease SI to introduce a few single-strand nicks and then treating with alkali as 1n Fig. lb. Bands 4 and 5 diminished and the bands of single-stranded plasmid DNA increased in intensity. This experiment allowed us to identify the position of "irreversibly denatured" CCC-DNA on gels, as well as to establish the approximate position of single- stranded plasmid DNA molecules. 1517 Downloaded from https://academic.oup.com/nar/article-abstract/7/6/1513/2380972/ by Ed 'DeepDyve' Gillespie user on 05 October 2019 Nucleic Acids Research If1 Figure 1. Alkaline treatment of plasmid DNA and cell extracts containing plasmid DMA. a-agarose gel electrophoresis of pBR322, purified by con- ventional methods, b- as in a, but treated with 0.1 N NaOH fo r 5 min. at room temperature before electrophoresis. c- as in b, but treated with nuclease SI for 15 m1n. before treatment with alkali ; d- as 1n c, but treated for 30 min. with SI. e- alkaline extract of E.coli HB101 cell s containing pBR322. This gel is overloaded to demonstrate the position of fluorescent bands other than the CCC-form of plasmid pBR322 (band 4). Band 1 - chromosomal DNA; band 2 - pBR322 dimer; band 3 - pBR322, OC-form; band 5 - "irreversibly denatured" form of pBR322 CCC DNA; band 6 - region of low molecular weight RNA. The alkaline extraction method was designed to prevent the generation of the "irreversibly denatured" form; at the same time, the extracts must be alkalin e enough for denaturation of chromosomal DNA to occur. Fig. le shows an example of such an extract, prepared according to the standard method. The gel is overloaded to indicate the relative positions of minor components. A 1S18 Downloaded from https://academic.oup.com/nar/article-abstract/7/6/1513/2380972/ by Ed 'DeepDyve' Gillespie user on 05 October 2019 Nucleic Acids Research small amount of the "irreversibly denatured" form (band 5) runs jus t ahead of the CCC form (band 4). Contaminating chromosomal DNA (band 1) and a large amount of low molecular weight RNA (band 6) are present. OC DNA (Band 3) and CCC-d1mer DNA (band 2) are also detectable. Since these other fluorescent bands may be present to varying extents during routine extractions of plasmid DNA, a knowledge of their relative mobilities 1s helpful in Interpreting gel electrophoresis patterns. Screening by size of recombinant plasmids prepared by the alkaline extraction method : A commonly used firs t step 1n the characterization of a new recombinant plasmid is determination of Its size. If i t is formed by integration of a fragment of foreign DNA, the size of the fragment may be estimated by agarose gel electrophoresis of either the Intact recombinant plasmid or the excised fragment. The alkaline extraction method permits both kinds of analyses. An example of the results of screening recombinant plasmids 1n a "shot-gun" type of experiment is shown in Fig. 2. Mouse DNA was cleaved Figure 2. Screening of mouse DNA/pBR322 recombinant plasmids by gel electro- phoresis of alkali-extracted plasmid DNA. r - reference mixture of CCC-DNAs; the sizes of reference plasmids (in order of decreasing nobility) are 4.3 kbp (pBR322), 5.7 kbp, 8.5 kbp and 11.2 kbp (pSF 2124). Minor bands are contam- inatin g OC forms in the reference mixture. 1519 Downloaded from https://academic.oup.com/nar/article-abstract/7/6/1513/2380972/ by Ed 'DeepDyve' Gillespie user on 05 October 2019 Nucleic Acids Research by Hind II I and Inserted into the single Hind II I site of pBR322. ApR TcS clones were selected and plasmid DNA extracted by the standard method. After agarose gel electrophoresis, the principal band seen 1n each slot has a mobility less than that of the pBR322 CCC-DNA used as a reference (fastest moving band i n slot r) . The estimated range of integrated DNA fragments, based upon mobilit y relative to marker CCC-DNAs, was from a few hundred bp up to 3 kbp. A band of plasmid DNA can be detected in each slot although, on a routine screening basis, the recovery of plasmid DNA varied somewhat. We estimate that, on the average, there 1s about 1 ug of plasmid DNA extracted from 1 ml of origina l non-amplified culture. Restriction enzyme digestion : A second Important method for characterizing recombinant plasmids 1s restriction enzyme digestion. We have found that the alkaline extraction procedure gives a preparation of plasmid DNA which 1s pure enough to be susceptible to digestion by several restriction enzymes including Eco RI, H1nd III , Bam HI , Hinf I , Ava II , H1nc III , and Pst I (data not shown). By eliminating the requirement for more highly purified DNA, thi s method should simplify characterization of recombinant plasmids. Transformation by extracted plasmid DNA : I t 1s often useful to be able to pass plasmid DNA from one cell to other bacterial cell strains or species. Extracts prepared according to the procedure we have described contain DNA suitable for use in transformation. Rapoport et al . (19) have used an earlier version of the method for this purpose and they found that 1n some cases a relativel y low efficiency of transformation was obtained. The yiel d of transformation has been Improved by the washing procedure described 1n Materials and Methods. We now fin d that extracts of pBR322 transform com- petent HB1O1 cell s at levels comparable to highly purified pBR322. Preparation of large plasmid DNAs : Recombinant plasmids, prepared by the "cosmid" technique (22), are expected to be 38-52 kbp 1n length. We tested our alkaline extraction method to see i f i t could be applied to plasmid DNAs in this size range since other methods employing alkali have been used successfully (20,21). E.coli DNA, partiall y digested with H1nd III , was inserted into plasmid pHC79 (from B. Hohn). In vitro packaging of the ligate d DNA and transduction into E.coli strain SF8 was carried out under the directio n of B. Hohn during an EMBO Course. Several Ap clones were selected and plasmid DNA was prepared from two 4-mm dia. colonies as described in Materials and Methods. The DNA was examined by agarose gel electrophoresis 1520 Downloaded from https://academic.oup.com/nar/article-abstract/7/6/1513/2380972/ by Ed 'DeepDyve' Gillespie user on 05 October 2019 Nucleic Acids Research eithe r directly (Fig. 3a and 3e), or after digestion with Hind II I (Fig. 3b and 3f) . Linear cosmid DNA, generated by digestion with H1nd III , migrates at a rate very close to that of the X 7.0 kbp fragment. The slowly migrating bands in 4a and 4e are presumed to be large CCC-DNAs. Digestion with H1nd II I generated the expected pHC79 sized fragment as well as several other fragments. Including some very large ones. Although digestion with restriction enzyme may not have been complete in this experiment, 1t 1s evident these large plasmids may be prepared by the alkaline extraction method. Preliminary results suggest that F'-plasmids (60-120 x 10 daltons) can also be extracted from appropriate strains of E.coli (kindly provided by R. d'Ari). CONCLUSIONS We have described a procedure for extracting partially purified Figure 3. Analysis of two "cosmid" DNAs prepared by the alkaline extraction method, a and e- undigested plasmid DNAs; b and f- digested with H1nd III; c- reference CCC-DNAs, 1n order of decreasing mobility: 4.3, 4.9, 5.7 and 11.2 kbp; d- reference linear DNA (Hind III digest of phage X DNA): 2.2, 2.5, 4.8, 7.0, 10.0 and 24.0 kbp. 1521 Downloaded from https://academic.oup.com/nar/article-abstract/7/6/1513/2380972/ by Ed 'DeepDyve' Gillespie user on 05 October 2019 Nucleic Acids Research plasmid DNA which we believe to be a useful addition to the lis t of other methods currently available. The method 1s simple and reliabl e and has been used successfully 1n several other laboratories without appreciable difficulty. Screening new recombinant plasmids by size is useful in initiall y characterizing new plasmids, 1n looking for plasmids containing inserts of a particular size, and perhaps 1n cases where no specific selection marker is available for detecting recombinants. Analysis of supercoiled DNA may give a better estimate of length than linear DNA fo r high molecular weight molecules (23). Plasmid DNA in this partially purified form is sensitive to digestion by those restrictio n enzymes which we have tested. This property of the extracts should simplif y analysis of recominant DNA molecules. ACKNOWLEDGEMENTS H.C.B. was supported i n part by a long-term EMBO fellowship . H.C.B. wishes to thank Dr. B. Bernardi, in whose laboratory thi s work was carrie d out , fo r his kind hospitality . We thank Dr. A. Kay fo r drawing referenc e 10 to out attention , Dr. A. Hinnen fo r providing a sample of yeast protoplasts , and Dr. B. Hohn for instructio n in "cosmid" preparation. Send correspondence to : H.C. Birnboim Atomic Energy o f Canada Limited Chalk River, Ontario KOJ UO (Canada). REFERENCES 1 . Clevell, D.B. and Helinski , D.R. (1969) Proc. Nat. Acad. Sci. USA 6g, 1159-1166. 2 . Colman, A. , Byers, M.J., Primrose, S.B. and Lyons, A. (1978) Eur. J . Biochem. 91., 303-310. 3. Barnes, W.M. (1977) Science 195, 393-394. 4. Telford, J., Boseley, P., SchaTfner, W. and Birnstiel, M. (1977) Science 195, 391-393. 5. EckTardt, T. (1978) Plasmid j. , 584-588. 6. Mickel, S., Arena, V. Jr. and Bauer, W. (1977) Nucleic Acids Research 4, 1465-1482. 7. Jansz, H.S., Pouwels, P.H., Schiphorst, J. (1966) Biochim. Biophys. Acta 123, 626-627. 8. Pouwels, P.H., Knijnenburg, CM. , van Rotterdam, J., Cohen, J.A. and Jansz, H.S. (1968) J. Mol. Biol. 32, 169-182. 9. Pouwels, P.H., van Rotterdam, J. ana" Cohen, J.A. (1968) J. Mol. Biol. 40_, 379-390. 10. Rush, M.G. and Warner, R.C. (1970) J . B1ol. Chem. 245_, 2704-2708. 11 . Kay, E.R.M., Simmons, N.S. and Dounce, A.L. (1952) J . Am. Chem. Soc. 74_, 1724-1726. 1522 Downloaded from https://academic.oup.com/nar/article-abstract/7/6/1513/2380972/ by Ed 'DeepDyve' Gillespie user on 05 October 2019 Nucleic Acids Research 12. Marko, A.M. and Butler , G.C. (1951) J. Biol . Chem. 190, 165-176. 13. Crestfield, A.M., Smith, K.C. and Allen , F.W. (19551 J . Biol. Chem. 216, 185-193. 14. Boyer, H.W. and Roulland-Dussoix (1969) J. Mol. B1ol. 41,, 459-472. 15. Kusner, S.R. (1978) in Genetic Engineering, Ed. H.W. Boyer and S. Nicosia, Elsevier/North-Holland , Biomedical Press, Amsterdam p. 17-23. 16. Bolivar, F., Rodriguez, R.L., Greene, P.J., Betlach, M.C., Heyneker, H.L. Boyer, H.L., Crosa, J.H. and Falkow, S. (1977) Gene 2, 95-113. 17. Clewell, D.B. (1972) J. Bacteriol. 110, 667-676. 18. Bedbrook, J.R. and Ausubel, F.M. (1976T Cell i , 707-716. 19. Rapoport, G. Klier , A., Billault , A., Fargette, F. and Dedonder, R. (1979) Molec. Gen. Genet., in press. 20- Sharp, P.A., Hsu, M.-T., Ohtsubo, E. and Davidson, N. (1972) J . Mol. Biol . 71, 471-497. 21 . Currier, T.C. an? Nester, E.W. (1976) Analyt. Biochem. 76, 431-441. 22 . Collins, J. and Hohn, B. (1978) Proc. Nat. Acad. Sci. U$A~ 75 , 4242-4246. 23. CoTlins, J . (1977) 1n Current Topics in Microbiology and Immunology, Springer-Verlag , Berlin, p. 121. 1523 Downloaded from https://academic.oup.com/nar/article-abstract/7/6/1513/2380972/ by Ed 'DeepDyve' Gillespie user on 05 October 2019 Nucleic Acids Research http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Nucleic Acids Research Oxford University Press

A rapid alkaline extraction procedure for screening recombinant plasmid DNA

Bimboim,, H.C.; Doly,, J.
Nucleic Acids Research , Volume 7 (6) – Nov 24, 1979
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Oxford University Press
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© 1979 Information Retrieval Limited
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0305-1048
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Abstract

Downloaded from https://academic.oup.com/nar/article-abstract/7/6/1513/2380972/ by Ed 'DeepDyve' Gillespie user on 05 October 2019 Volume 7 Number 61979 Nucleic Acid s Research H.C.Bimboim+ and J.Doly Laboratoire de Ge'ne'tique Moleculaire, Institut de Recherche en Biologie Moleculaire, F-75221 Paris Cedex 05, France Received 3 August 1979 ABSTRACT A procedure for extracting plasmid DNA from bacterial cells 1s described. The method 1s simple enough to permit the analysis by gel electrophoresis of 100 or more clones per day yet yields plasmid DNA which is pure enough to be digestible by restriction enzymes. The principle of the method is selective alkaline denaturation of high molecular weight chromosomal DNA while covalently closed circular DNA remains double-stranded. Adequate pH control is accomplished without using a pH meter. Upon neutralization, chromosomal DNA renatures to form an insoluble clot, leaving plasmid DNA in the supernatant. Large and small plasmid DNAs have been extracted by this method. INTRODUCTION Bacterial plasmid DNAs are widely used as cloning vehicles in re- combinant DNA research. After new plasmids are constructed, they may be isolated and characterized with respect to their size and restriction enzyme pattern by gel electrophoresis. One method for preparing plasmid DNA In a highly purified form involves gently lysis of bacterial cells, centrifugation to remove the bulk of the chromosomal DNA, and then banding of residual DNA in a cesium chloride gradient in the presence of ethidium bomide. Covalently closed circular (CCC) DNA binds a different amount of the dye than does open circular (OC) or linear DNA and is readily separated from the latter two forms of DNA (1). A second, more rapid, method for preparing plasmid DNA employs hydroxyapatite chromatography (2); this also produces highly purified DNA. However, for many purposes including analysis by gel electrophoresis, less purified DNA can be used. Under favourable conditions, a colony of cells can be lysed and plasmid DNA can be detected in crude extracts after electro- phoresis (3-6); this permits the analysis of many clones and makes screening possible. In this report we describe another method for plasmid DNA extraction which is both simple enough to permit screening of many small samples, yet O Information Retrieval Limited 1 Falconberg Court London W1V5F G England 1613 Downloaded from https://academic.oup.com/nar/article-abstract/7/6/1513/2380972/ by Ed 'DeepDyve' Gillespie user on 05 October 2019 Nucleic Acids Research yield s plasnrid DNA in a form sufficientl y pure to be digestible by restriction enzymes or to be used to transform other cells. In these respects, the pro- cedure we describe is more versatile than other rapid extraction methods. Plasmid DNA can be detected in as littl e as 0.1 ml of non-amplified liquid cultur e or 1n a colony of cells scraped from a plate. PRINCIPLE OF THE ALKALINE EXTRACTION METHOD Previous workers have shown that there is a narrow range of pH (about 12.0-12.5) within which denaturation of linear DNA but not CCC-DNA occurs and that this property can be used for purifying CCC-DNA (7-10,20,21). We have utilize d this approach for developing a rapid extraction method for plasmid DNAs. Plasmid-containing cells are treated with lysozyme to weaken the cell wall and then lysed completely with sodium dodecyl sulfate (SDS) and NaOH. By choosing the ratio of cell suspension to NaOH solutio n carefully, a reproducible alkaline pH value 1s obtained without the necessity of monitoring the pH wit h a meter; further pH control is obtained by including glucose as a pH buffer. Chromosomal DNA, stil l in a very high molecular weight form, 1s selectivel y denatured ?nd when the lysate is neutralized by acidic sodium acetate, the mass of chromosomal DNA renatures and aggregates to form an insoluble network. Simultaneously, the high concentration of sodium acetate causes precipitation of protein-SDS complexes (11,12) and of high molecular weight RNA (13). In this way, most of the three major contaminating macro- molecules are co-precipitated and may be removed by a single centrifugation i n a bench-top centrifuge. Plasmid DNA (and residual low molecular weight RNA) are recovered from the supernatant by ethanol precipitation. Plasmid DNA may be analyzed by gel electrophoresis either intact in the CCC form or after digestion with a restrictio n enzyme. MATERIALS AND METHODS Cell strains and Media : Escherichia coli strains HB101 (14), SK1592 (15) and RRI (16) were used for most of the work reported here. E.coli F' strains GM218 (thr" , leu", arg", 1lv", thy", ura", his" , recA"/F'h1s+) and JM856 (thyA", lysA", pheA", arg", relA", recA'/F1 thy , lys , phe , arg , relA , tif ) from J . George were grown in minimal medium supplemented with the appro- priat e nutritional requirements at 30°C. Otherwise, L-broth was used through- out . When appropriate, Ampicillin (Ap) was added at a concentration of 100 ug/ml for liquid medium or 40 ug/ml for plates. Tetracycline (Tc) was used 1514 Downloaded from https://academic.oup.com/nar/article-abstract/7/6/1513/2380972/ by Ed 'DeepDyve' Gillespie user on 05 October 2019 Nucleic Acids Research fo r agar plates at a concentration of 10 ug/ml. Equipment and Reagents for Plasmid Extraction : Eppendorf-type (1.5 ml) polypropylene tubes and a bench-top centrifuge capable of generating 8-10,000 x g were used. A rack holding 60 Eppendorf tubes speeds up handling of larger numbers of samples. Several racks can be prepared by stacking 5 sheets of 2 nrn-thick aluminum (or other metal) and drillin g 11 mm-d1a. holes at 15 mm spacing through all simultaneously; the edges of each sheet are folded as legs. For complete removal of supernatants following centrifugation in Eppendorf tubes, a Pasteur pipette drawn out to a fine tip is used. Reagents : I. Lysozyme solution - 2 mg/ml lysozyme, 50 mM glucose, 10 mM CDTA, 25 mM Tris-HC l (pH 8.0). Prepare fresh daily from crystalline lysozyme and stock solutions of the other components. Store at 0°C. II . Alka- lin e SDS solution - 0.2 N NaOH, IS sodium dodecyl sulfate (SDS). Store at room temperature; stable for about 1 week. III. High salt solution - 3 M sodium acetate (pH 4.8). Prepare by dissolving 3 moles of sodium acetate in a minimal volume of water, adjusting to pH 4.8 with glacial acetic acid, and then adjusting volume to 1 1. Store at room temperature. CDTA (cyclohexane diamine tetracetate) 1s a chelating agent which is more soluble in alcohol and forms stronger complexes with metal ions than does EDTA. EDTA can be substituted fo r applications listed here. CDTA from Sigma or Aldrich Chemical Company was used. A stock solution of RNAse A (1 mg/ml in 5 mM Tris-HCl , pH 8.0) is treated by heating at 100° for 10 m1n. Standard Procedure for Extraction of Plasmid DNA from Small Volumes of Cell Cultures : (Footnotes refer to comments below.) The method has been used principally with plasmid pBR322 (ref. 16) and it s derivatives in E.coli strains HB101, RRI and SK 15921. Selected clones (ApR, TcS in the case of hybrid plasmids with inserts at the H1nd II I site of pBR322) are grown in 2.5 ml of L-broth containing 100 ug/ml Ap in 6-ml vials . After 18 h incubation, 0.5 ml of culture is transferred to a 1.5 ml Eppendorf tube for plasmid extraction , and the remainder is stored at -20°C after the addition of glycerol to 40J. All manipulations are carried out at room temperature unless otherwise Indicated. The tube is centrifuged for 15 seconds . The super- natant is carefully removed with a fine-ti p aspirator and the cell pellet is thoroughly suspended 1n 100 ul of solution I. After a 30 min. period of incubation at 0°C, 200 ml of solution I I 1s added and the tube is gently vortexed. The suspension should become almost clear and slightl y viscous. The tube is maintained for 5 min. at 0°C and then 150 ul of solution II I is 1515 Downloaded from https://academic.oup.com/nar/article-abstract/7/6/1513/2380972/ by Ed 'DeepDyve' Gillespie user on 05 October 2019 Nucleic Acids Research added. The contents of the tube are gently mixed by inversion for a few seconds during which time a clot of DNA forms. The tube is maintained at 0°C fo r 60 min. to allow most of the protein, high molecular weight RNA and chro- mosomal DNA to precipitate. Centrifugation for 5 m1n. yields an almost clear supernatant. Four-tenths of a ml of the supernatant is removed and trans- ferred to a second centrifuge tube . Small amounts of floating material may be carried over at this time. One ml of cold ethanol is added and the tube i s held at -20°C for 30 min. The precipitate 1s collected by centrifugation fo r 2 m1n. and the supernatant removed by aspiration. The pellet 1s dissolved 1n 100 ul of 0.1 M sodium acetate/0.05 M Tris-HCl (pH 8) and repredpitated with 2 volumes of cold ethanol. After 10 min. at -20°C, the precipitate is again collected by centrifugation as before. The pellet 1s dissolved in 40 ul water and then 10 ul of 5 x sample buffer 1s added. 10-20 ul is applied to an agarose gel for electrophoretic analysis. Modifications required when plasmid DNA is to be used for bacterial transformation or is to be treated by restriction enzymes : The pellet at the las t step is redissolved once more 1n 100 ul of 0.1 M sodium acetate/0.05 M Tris-HCl (pH 8) Instead of water and precipitated with 2 volumes of ethanol as before. For transformation, the pellet may be dissolved in 40 ul of water or dilute buffer and a few microliters added to competent cells. For restric- tio n enzyme analysis, the pellet 1s dissolved in 36 ul hLO and 4 ul of pan- creatic RNase (1 mg/ml) 1s added. After 30 min. incubation at 37°C, concen- trate d restriction enzyme buffer and 1 unit of enzyme are added. Following a period of digestion (37°, 60 min.), the sample is mixed with concentrated sample buffer and part of i t applied to a gel for electrophoretic analysis. Comments and other modifications of the method : 1. CCC-DNA in other cell types has been successfully extracted by the above method. Incubation at room temperature or 37°C with solution I may be necessary to disrupt Bacillus subtilis (S.D. Ehrlich, G. Rapoport - personal communication). A band corresponding in size to the 2 u circl e of yeast cells was detected by gel electrophoresis when alkaline extracts of Saccharomyces cerevisiae strain GRF18 protoplasts (kindly prepared by A. Hinnen) were examined. 2. One-half ml cultures have been grown directly in 1.5 ml Eppendorf tube ; alternatively, colonies (3-4 mm dia. ) can be scraped from the surface of an agar plate and resuspended in 100 ul of solution I. 3. If the cell number is very low, a longer centrifugation time may be required for the pellet to stick adequately to the wall of the tube or 0.2 ml of carrier cells can be added. 4. If the 1516 Downloaded from https://academic.oup.com/nar/article-abstract/7/6/1513/2380972/ by Ed 'DeepDyve' Gillespie user on 05 October 2019 Nucleic Acids Research cel l number is low, 25 ug of tRNA can be added as carrier to assist precipi- tatio n of plasmid DNA. Gel electrophoresis : 0.81 agarose gels were used, either as 6-tnn d1a. tubes or as 3-mm thick vertical slabs. Electrophoresis buffer contained 40 mM Tris , 20 mM sodium acetate, 2 mM EDTA, adjusted to pH 7.8 with acetic acid . 5 x sample buffer contained 25% sucrose, 5 mM sodium acetate, 0.05% bromophenol blue, 0.1% SDS. After electrophoresis, gels were stained with ethidium bromide (1 ug/ml) and photographed under UV illumination . Restrictio n Enzymes : Most were obtained from Biolabs and used under conditions described by the manufacturer. Hind II I was prepared at this institut e by A. Meier. Al l experiments Involving recombinant DNA were carried out with the permission of the French Commission on Recombinant DNA according to the French Guidelines. RESULTS AND DISCUSSION Treatment of plasmid DNA wit h alkali : The extraction method we have described involves exposing a crude extract to alkaline pH to denature chro- mosomal DNA. However, when covalently closed circular DNA is exposed to > pH 13, i t gives rise to a denatured form which is not readily renaturable (8,9) . The generation of this "irreversibly denatured" form (which is to be avoided in preparing plasmid DNA) is illustrated in Fig. 1. Fig. la shows the electrophoretic mobility of pBR322 DNA, purified by conventional methods (1). Band 4 is the supercoiled (CCC form). A small amount of 0C form (band 3) and CCC-dimer (band 2) are also visible. When treated with 0.1 N NaOH, neutralized, and run on a neutral gel, a faster moving band (band 5), corresponding to the "irreversibl y denatured" form, can be seen (Fig. lb). Two additional minor bands which migrate close to bands 4 and 5 are also seen. It is likely that these represent two single-stranded forms of plasmid DNA (linear and circular) which arise by denaturation of the 0C form. This was verified (Fig. lc and Id) by treating the CCC form with nuclease SI to introduce a few single-strand nicks and then treating with alkali as 1n Fig. lb. Bands 4 and 5 diminished and the bands of single-stranded plasmid DNA increased in intensity. This experiment allowed us to identify the position of "irreversibly denatured" CCC-DNA on gels, as well as to establish the approximate position of single- stranded plasmid DNA molecules. 1517 Downloaded from https://academic.oup.com/nar/article-abstract/7/6/1513/2380972/ by Ed 'DeepDyve' Gillespie user on 05 October 2019 Nucleic Acids Research If1 Figure 1. Alkaline treatment of plasmid DNA and cell extracts containing plasmid DMA. a-agarose gel electrophoresis of pBR322, purified by con- ventional methods, b- as in a, but treated with 0.1 N NaOH fo r 5 min. at room temperature before electrophoresis. c- as in b, but treated with nuclease SI for 15 m1n. before treatment with alkali ; d- as 1n c, but treated for 30 min. with SI. e- alkaline extract of E.coli HB101 cell s containing pBR322. This gel is overloaded to demonstrate the position of fluorescent bands other than the CCC-form of plasmid pBR322 (band 4). Band 1 - chromosomal DNA; band 2 - pBR322 dimer; band 3 - pBR322, OC-form; band 5 - "irreversibly denatured" form of pBR322 CCC DNA; band 6 - region of low molecular weight RNA. The alkaline extraction method was designed to prevent the generation of the "irreversibly denatured" form; at the same time, the extracts must be alkalin e enough for denaturation of chromosomal DNA to occur. Fig. le shows an example of such an extract, prepared according to the standard method. The gel is overloaded to indicate the relative positions of minor components. A 1S18 Downloaded from https://academic.oup.com/nar/article-abstract/7/6/1513/2380972/ by Ed 'DeepDyve' Gillespie user on 05 October 2019 Nucleic Acids Research small amount of the "irreversibly denatured" form (band 5) runs jus t ahead of the CCC form (band 4). Contaminating chromosomal DNA (band 1) and a large amount of low molecular weight RNA (band 6) are present. OC DNA (Band 3) and CCC-d1mer DNA (band 2) are also detectable. Since these other fluorescent bands may be present to varying extents during routine extractions of plasmid DNA, a knowledge of their relative mobilities 1s helpful in Interpreting gel electrophoresis patterns. Screening by size of recombinant plasmids prepared by the alkaline extraction method : A commonly used firs t step 1n the characterization of a new recombinant plasmid is determination of Its size. If i t is formed by integration of a fragment of foreign DNA, the size of the fragment may be estimated by agarose gel electrophoresis of either the Intact recombinant plasmid or the excised fragment. The alkaline extraction method permits both kinds of analyses. An example of the results of screening recombinant plasmids 1n a "shot-gun" type of experiment is shown in Fig. 2. Mouse DNA was cleaved Figure 2. Screening of mouse DNA/pBR322 recombinant plasmids by gel electro- phoresis of alkali-extracted plasmid DNA. r - reference mixture of CCC-DNAs; the sizes of reference plasmids (in order of decreasing nobility) are 4.3 kbp (pBR322), 5.7 kbp, 8.5 kbp and 11.2 kbp (pSF 2124). Minor bands are contam- inatin g OC forms in the reference mixture. 1519 Downloaded from https://academic.oup.com/nar/article-abstract/7/6/1513/2380972/ by Ed 'DeepDyve' Gillespie user on 05 October 2019 Nucleic Acids Research by Hind II I and Inserted into the single Hind II I site of pBR322. ApR TcS clones were selected and plasmid DNA extracted by the standard method. After agarose gel electrophoresis, the principal band seen 1n each slot has a mobility less than that of the pBR322 CCC-DNA used as a reference (fastest moving band i n slot r) . The estimated range of integrated DNA fragments, based upon mobilit y relative to marker CCC-DNAs, was from a few hundred bp up to 3 kbp. A band of plasmid DNA can be detected in each slot although, on a routine screening basis, the recovery of plasmid DNA varied somewhat. We estimate that, on the average, there 1s about 1 ug of plasmid DNA extracted from 1 ml of origina l non-amplified culture. Restriction enzyme digestion : A second Important method for characterizing recombinant plasmids 1s restriction enzyme digestion. We have found that the alkaline extraction procedure gives a preparation of plasmid DNA which 1s pure enough to be susceptible to digestion by several restriction enzymes including Eco RI, H1nd III , Bam HI , Hinf I , Ava II , H1nc III , and Pst I (data not shown). By eliminating the requirement for more highly purified DNA, thi s method should simplify characterization of recombinant plasmids. Transformation by extracted plasmid DNA : I t 1s often useful to be able to pass plasmid DNA from one cell to other bacterial cell strains or species. Extracts prepared according to the procedure we have described contain DNA suitable for use in transformation. Rapoport et al . (19) have used an earlier version of the method for this purpose and they found that 1n some cases a relativel y low efficiency of transformation was obtained. The yiel d of transformation has been Improved by the washing procedure described 1n Materials and Methods. We now fin d that extracts of pBR322 transform com- petent HB1O1 cell s at levels comparable to highly purified pBR322. Preparation of large plasmid DNAs : Recombinant plasmids, prepared by the "cosmid" technique (22), are expected to be 38-52 kbp 1n length. We tested our alkaline extraction method to see i f i t could be applied to plasmid DNAs in this size range since other methods employing alkali have been used successfully (20,21). E.coli DNA, partiall y digested with H1nd III , was inserted into plasmid pHC79 (from B. Hohn). In vitro packaging of the ligate d DNA and transduction into E.coli strain SF8 was carried out under the directio n of B. Hohn during an EMBO Course. Several Ap clones were selected and plasmid DNA was prepared from two 4-mm dia. colonies as described in Materials and Methods. The DNA was examined by agarose gel electrophoresis 1520 Downloaded from https://academic.oup.com/nar/article-abstract/7/6/1513/2380972/ by Ed 'DeepDyve' Gillespie user on 05 October 2019 Nucleic Acids Research eithe r directly (Fig. 3a and 3e), or after digestion with Hind II I (Fig. 3b and 3f) . Linear cosmid DNA, generated by digestion with H1nd III , migrates at a rate very close to that of the X 7.0 kbp fragment. The slowly migrating bands in 4a and 4e are presumed to be large CCC-DNAs. Digestion with H1nd II I generated the expected pHC79 sized fragment as well as several other fragments. Including some very large ones. Although digestion with restriction enzyme may not have been complete in this experiment, 1t 1s evident these large plasmids may be prepared by the alkaline extraction method. Preliminary results suggest that F'-plasmids (60-120 x 10 daltons) can also be extracted from appropriate strains of E.coli (kindly provided by R. d'Ari). CONCLUSIONS We have described a procedure for extracting partially purified Figure 3. Analysis of two "cosmid" DNAs prepared by the alkaline extraction method, a and e- undigested plasmid DNAs; b and f- digested with H1nd III; c- reference CCC-DNAs, 1n order of decreasing mobility: 4.3, 4.9, 5.7 and 11.2 kbp; d- reference linear DNA (Hind III digest of phage X DNA): 2.2, 2.5, 4.8, 7.0, 10.0 and 24.0 kbp. 1521 Downloaded from https://academic.oup.com/nar/article-abstract/7/6/1513/2380972/ by Ed 'DeepDyve' Gillespie user on 05 October 2019 Nucleic Acids Research plasmid DNA which we believe to be a useful addition to the lis t of other methods currently available. The method 1s simple and reliabl e and has been used successfully 1n several other laboratories without appreciable difficulty. Screening new recombinant plasmids by size is useful in initiall y characterizing new plasmids, 1n looking for plasmids containing inserts of a particular size, and perhaps 1n cases where no specific selection marker is available for detecting recombinants. Analysis of supercoiled DNA may give a better estimate of length than linear DNA fo r high molecular weight molecules (23). Plasmid DNA in this partially purified form is sensitive to digestion by those restrictio n enzymes which we have tested. This property of the extracts should simplif y analysis of recominant DNA molecules. ACKNOWLEDGEMENTS H.C.B. was supported i n part by a long-term EMBO fellowship . H.C.B. wishes to thank Dr. B. Bernardi, in whose laboratory thi s work was carrie d out , fo r his kind hospitality . We thank Dr. A. Kay fo r drawing referenc e 10 to out attention , Dr. A. Hinnen fo r providing a sample of yeast protoplasts , and Dr. B. Hohn for instructio n in "cosmid" preparation. Send correspondence to : H.C. Birnboim Atomic Energy o f Canada Limited Chalk River, Ontario KOJ UO (Canada). REFERENCES 1 . Clevell, D.B. and Helinski , D.R. (1969) Proc. Nat. Acad. Sci. USA 6g, 1159-1166. 2 . Colman, A. , Byers, M.J., Primrose, S.B. and Lyons, A. (1978) Eur. J . Biochem. 91., 303-310. 3. Barnes, W.M. (1977) Science 195, 393-394. 4. Telford, J., Boseley, P., SchaTfner, W. and Birnstiel, M. (1977) Science 195, 391-393. 5. EckTardt, T. (1978) Plasmid j. , 584-588. 6. Mickel, S., Arena, V. Jr. and Bauer, W. (1977) Nucleic Acids Research 4, 1465-1482. 7. Jansz, H.S., Pouwels, P.H., Schiphorst, J. (1966) Biochim. Biophys. Acta 123, 626-627. 8. Pouwels, P.H., Knijnenburg, CM. , van Rotterdam, J., Cohen, J.A. and Jansz, H.S. (1968) J. Mol. Biol. 32, 169-182. 9. Pouwels, P.H., van Rotterdam, J. ana" Cohen, J.A. (1968) J. Mol. Biol. 40_, 379-390. 10. Rush, M.G. and Warner, R.C. (1970) J . B1ol. Chem. 245_, 2704-2708. 11 . Kay, E.R.M., Simmons, N.S. and Dounce, A.L. (1952) J . Am. Chem. Soc. 74_, 1724-1726. 1522 Downloaded from https://academic.oup.com/nar/article-abstract/7/6/1513/2380972/ by Ed 'DeepDyve' Gillespie user on 05 October 2019 Nucleic Acids Research 12. Marko, A.M. and Butler , G.C. (1951) J. Biol . Chem. 190, 165-176. 13. Crestfield, A.M., Smith, K.C. and Allen , F.W. (19551 J . Biol. Chem. 216, 185-193. 14. Boyer, H.W. and Roulland-Dussoix (1969) J. Mol. B1ol. 41,, 459-472. 15. Kusner, S.R. (1978) in Genetic Engineering, Ed. H.W. Boyer and S. Nicosia, Elsevier/North-Holland , Biomedical Press, Amsterdam p. 17-23. 16. Bolivar, F., Rodriguez, R.L., Greene, P.J., Betlach, M.C., Heyneker, H.L. Boyer, H.L., Crosa, J.H. and Falkow, S. (1977) Gene 2, 95-113. 17. Clewell, D.B. (1972) J. Bacteriol. 110, 667-676. 18. Bedbrook, J.R. and Ausubel, F.M. (1976T Cell i , 707-716. 19. Rapoport, G. Klier , A., Billault , A., Fargette, F. and Dedonder, R. (1979) Molec. Gen. Genet., in press. 20- Sharp, P.A., Hsu, M.-T., Ohtsubo, E. and Davidson, N. (1972) J . Mol. Biol . 71, 471-497. 21 . Currier, T.C. an? Nester, E.W. (1976) Analyt. Biochem. 76, 431-441. 22 . Collins, J. and Hohn, B. (1978) Proc. Nat. Acad. Sci. U$A~ 75 , 4242-4246. 23. CoTlins, J . (1977) 1n Current Topics in Microbiology and Immunology, Springer-Verlag , Berlin, p. 121. 1523 Downloaded from https://academic.oup.com/nar/article-abstract/7/6/1513/2380972/ by Ed 'DeepDyve' Gillespie user on 05 October 2019 Nucleic Acids Research

Journal

Nucleic Acids Research – Oxford University Press

Published: Nov 24, 1979

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