ANALYTICAL BIOCHEMISTRY 121, 382-387 (1982)

A Procedure for the Large-Scale isolation of Highly Purified Plasmid DNA Using Alkaline Extraction and Binding to Glass Powder

M. A. MARKO,' R. CHIPPERFIELD, AND H. C. BIRNBOIM,'

Radiation Biology Branch, Health Sciences Division. Chalk River Nuclear Laboratories, Chalk River, Ontario KOJ I JO. Canada

Received October 19, 1981

A preparative procedure for obtaining highly purified plasmid DNA from bacterial cells is described. The method is adapted from our earlier procedure, which gave partially purified plasmid in a form suitable for rapid screening of a large number of samples. In the present method, all detectable RNA, chromosomal DNA, and protein are removed without the use of enzymes, phenol extraction, dialysis, or equilibrium centrifugation. Binding of plasmid DNA to glass powder in the presence of 6 M sodium perchlorate is used for the final purification step.

The development of recombinant DNA research during the past decade has stimu- lated investigations into the development of new techniques for the isolation of purified bacterial plasmid DNAs. At present, a widely used method is to band the DNA from a “cleared lysate” in a cesium chloride gradient in the presence of ethidium bro- mide; covalently closed circular (CCC)3 DNA is separated from open circular (OC) and chromosomal DNA by this procedure ( 1). Other methods employ chromatography on hydroxyapatite (2) or acridine yellow gel (3).

In this report we describe an extension of an earlier analytical procedure (4), which allows isolation of highly purified plasmid DNA in large quantities without the use of cesium chloride banding, ribonuclease treat-

’ Present address: Department of Microbiology, Sun- nybrook Medical Centre, 2075 Bayview Ave., Toronto, Ontario, M4N 3M5 Canada.

’ To whom correspondence should be directed. ’ Abbreviations used: CCC, covalently closed circu-

lar; OC, open circular; CDTA, cyclohexanediamine tet- raacetate; DABA, 3,5-diaminobenzoic acid dihydro- chloride; BSA, bovine serum albumin; PEB, plasmid extraction buffer; SDS, sodium dodecyl sulfate; LB, loading buffer; EB, elution buffer.

ment, phenol extraction, or dialysis. Extrac- tion of lysozyme-treated bacterial cells un- der defined alkaline conditions selectively denatures chromosomal DNA but not CCC- plasmid DNA. When the crude alkaline ex- tract is neutralized, high-molecular-weight chromosomal DNA aggregates to form an insoluble network; high-molecular-weight RNA and protein-SDS complexes are like- wise rendered insoluble by the simultaneous addition of a high concentration of salt. Af- ter removal of insoluble material by centrif- ugation, soluble plasmid DNA is bound to glass powder (5-7) and washed extensively with sodium perchlorate to remove remain- ing contaminants. Finally, highly purified DNA is recovered in high yield by elution with a low-ionic-strength buffer.

MATERIALS AND METHODS

Chemicals and Enzymes

Ampicillin and lysozyme (egg white, grade I) were from Sigma Chemical Company, St. Louis, Missouri. CDTA (cyclohexanedi- amine tetraacetate) and DABA (3,5-diami- nobenzoic acid dihydrochloride) were from Aldrich Chemical Company, Milwaukee,

0003-2697/82/060382-06$02.00/O Copyright 0 1982 by Academic Press, Inc. All rights of reproduction in any form reserved.

382

ISOLATION OF PURIFIED PLASMID DNA 383

Wisconsin. DABA was further purified ac- cording to (8). BSA (bovine serum albumin, A grade), chloramphenicol (B grade), ri- bonuclease (from bovine pancreas, A grade), and ribonuclease T, were supplied by Cal- biochem-Behring Corporation, San Diego, California. Fluorescamine was from Roche Diagnostics, Nutley, New Jersey. Proteinase K was from E. Merck, Darmstadt, Federal Republic of Germany, and restriction en- donuclease Hind111 was from Boehringer- Mannheim Canada, Dorval, Province of Quebec.

Plasmid Extraction Buffers

PEB I: 50 mM glucose, 25 mM Tris-HCl, 10 mM CDTA (pH 8.0). PEB II: 0.1 N

NaOH, 1.0% sodium dodecyl sulfate. PEB III: 3 M sodium acetate (adjusted to pH 6.0 with glacial acetic acid). These solutions are modified slightly from those used in our ear- lier method (4). Loading buffer (LB): 6 M

NaClO,, 50 mM Tris-HCI, 10 mM CDTA (pH 8.0). Elution buffer (EB): 0.2 M NaClQ,, 60 mrvr Tris-HCl, 10 mM CDTA.

Preparation of Glass Powder

Glass-fiber filters (Whatman GF/A, lo- cm diameter) were ground with a mortar and pestle to a fine powder. The powder was suspended in water (50 ml/g of powder) and allowed to settle overnight before decanting off the fines. The glass powder was equili- brated with LB by pouring a slurry onto a 0.45-pm Millipore filter and washing with 10 ml of LB per gram of glass under gentle suction. A stock suspension of glass powder was prepared ( 1 g to 10 ml of LB) and stored at room temperature.

Detailed Procedure for Isolation of Plasmid pBR322 DNA from Escherichia coli

( 1) Growth of cells. Escherichia coli strain RR1 containing pBR322 (9) as a di- mer was grown overnight as a preculture in

20 ml of L-broth containing 100 pg/ml of ampicillin at 37°C. For amplified cells, 10 ml of preculture was used to inoculate 1 liter of L-broth containing ampicillin and incu- bated with vigorous shaking at 37°C until the ODbm was 0.98. Chloramphenicol was then added to a final concentration of 170 pg/ml for plasmid amplification and the in- cubation was continued for approximately 16 h ( 1). For nonamplified cells, 1 liter of L-broth with ampicillin was inoculated with the same preculture. The cells were grown for 24 h with vigorous shaking at 37°C (final ODem was 4.0).

(2) Lysis and the first alkali treatment. Amplified cells were collected by centrifu- gation and washed once with cold water. The cells from 1 liter of culture were resuspended thoroughly in 1.0 ml of PEB I at 0°C and transferred to a 125-ml Erlenmeyer flask. The centrifuge tubes were rinsed with 9.0 ml of PEB I containing 10 mg of lysozyme and this was combined. After mixing, the cell suspension was allowed to stand for 30 min at 0°C. Twenty milliliters of PEB II (at room temperature) was then added to lyse cells and denature chromosomal DNA; the viscous mixture was gently stirred with a glass rod until it became fairly clear and homogenous. After standing for 10 min at room temperature, 15 ml of PEB III (at room temperature) was added; this mixture was stirred with a glass rod until the viscosity of the solution markedly decreased and a coarse precipitate containing chromosomal DNA formed. After 1 h at 0°C the sample was centrifuged at 15,000g for 20 min at 0°C. The supernatant was mixed with 2 vol of cold ethanol and, after 1 h at -20°C was centrifuged at 16,OOOg for 20 min at - 10°C.

The procedure for nonamplified cultures was the same as that for amplified cultures, except that the volume of reagents used was twice that described above. From this point on, all steps were identical for amplified and nonamplified preparations.

(3) Second alkali treatment to remove residual chromosomal DNA (optional). The

384 MARKO, CHIPPERFIELD. AND BIRNBOIM

ethanol precipitate was dissolved in 2.0 ml of PEB I and 4.0 ml of PEB II was added. After 5 min at room temperature, 3.0 ml of PEB III was added and the mixture allowed to stand for 30 min at room temperature. The insoluble material was removed by cen- trifugation at 12,000g for 10 min at 25°C. Plasmid DNA was recovered from the clar- ified supernatant by centrifugation after pre- cipitation with 2 vol of cold ethanol.

(4) 5 M Lithium chloride treatment to remove residual ribosomal RNA and single- stranded DNA. The pellet was dissolved in 2.67 ml of 50 mM Tris-HCI, 1.5 mM CDTA (pH 8.0); 1.33 ml of 15 LiCl, 50 mM Tris- HCl (pH 8.0) was added and the mixture allowed to stand for 15 min at room tem- perature. The resulting precipitate was re- moved by centrifugation at 12,000g for 10 min at 4°C and plasmid DNA was recovered from the supernatant by ethanol precipita- tion.

(5) Proteinase K digestion (optional). The pellet was dissolved in 2.0 ml of 0.2% SDS, 10 mM Tris-HCl, 10 mM CDTA (pH 8.0). Forty microliters of proteinase K (1 mg/ml) was added and the sample was in- cubated for 30 min at 45°C. Plasmid DNA was recovered by centrifugation after addi- tion of sodium acetate to 0.1 M and 2 vol of ethanol.

(6) Final purification by adsorption to glass powder. The pellet was dissolved in 2.0 ml of LB, combined with 20 ml of a stock suspension of glass powder, and gently mixed for 15 min at room temperature. The slurry was poured onto a 0.45~pm Millipore HA filter (47-mm diameter) supported in a sin- tered glass holder and gentle suction was applied. The flow-through was collected and set aside to verify that the capacity of the glass for plasmid DNA had not been ex- ceeded. The glass powder was washed with 120 ml of LB, care being taken not to allow the powder to dry. DNA was eluted with 24 ml of EB under gentle suction at a rate of l-2 ml/min; all interstitial liquid was re- covered by applying full suction. The volume

recovered was about 30 ml. Purified plasmid DNA was concentrated by precipitation with 2 vol of ethanol. The pellet was redis- solved in 4.0 ml of 0.1 M sodium acetate, 50 mM Tris-HCI (pH 8.0) and reprecipitated with ethanol. The final pellet was dissolved in 1.0 ml of 5 mM Tris-HCl, 0.1 mM CDTA (pH 8.0) and stored at 4°C over a drop of chloroform.

Steps 3 and 5 are optional and may be omitted when small amounts of contami- nating chromosomal DNA or protein can be tolerated.

Quantitation of DNA

Purified plasmid DNA was quantitated by absorbance (1 AzbO unit = 50 pg). Impure preparations were quantitated fluorometri- tally with DABA (8), using pure plasmid DNA for preparing a standard curve.

Analysis for RNA Contamination in Purified Plasmid DNA

Low levels of RNA contamination in preparations of DNA are difficult to detect. In particular, if ribonuclease is used, oligo- nucleotides may be generated which are difficult to remove because some may be ethanol-precipitable, nondialyzable, and nonsedimentable in cesium chloride gra- dients. Furthermore, small oligonucleotides do not stain with ethidium bromide in aga- rose gels. The procedure we have described removed RNA without using ribonuclease. To verify that RNA was eliminated, a DNA sample was treated with NaOH to degrade RNA into mononucleotides (10) and the lat- ter were separated from high-molecular- weight DNA by chromatography on a Seph- adex column (11). Plasmid DNA (10 pg) was incubated at 37°C for 16 h with 0.2 N

NaOH. After neutralization with HCl, the sample was chromatographed on Sephadex G-50 fine (0.9 X 23 cm) in buffer containing 0.1 M NaCl, 10 mM Tris-HCl, 10 mM CDTA

ISOLATION OF PURIFIED PLASMID DNA 385

(pH 8.0) at 1.0 ml/min. The effluent was RESULTS AND DISCUSSION monitored at 254 nm with a Chromatronix Model 220 flow detector. The estimated de- tection limit when 10 pg of DNA was ap- plied was 0.3 pg of ribonucleotide material.

Analysis for Protein Contamination

Fluorescamine was used for detecting pro- teins, with BSA as a reference protein. DNA (ethanol-precipitated to remove Tris) in 0.5 ml of H,O was mixed with 1.0 ml of 0.3 M

sodium borate, 0.15% Triton X-100 (pH 9.45). One-half milliliter of fluorescamine (15 mg in 100 ml of acetone) was added with mixing. After addition of 0.5 ml of H20, the fluorescence was determined (Ex 390 nm; An 475 nm).

Monitoring of DNA Purification by Agarose Gel Electrophoresis

Alkali, treatment of lysozyme-treated bac- terial cells containing plasmid pBR322 leads to lysis and selective denaturation of chro- mosomal DNA; on neutralizing, chromo- somal DNA renatures to form an insoluble aggregate, leaving plasmid DNA in solution. Our earlier analytical procedure (4) yields plasmid DNA which is sufficiently pure for many purposes, but it still contains some residual chromosomal DNA, RNA, and pro- tein. The present protocol was developed to remove all traces of these contaminants. At each stage in purification, an aliquot was taken and analyzed by gel electrophoresis (Fig. 1 for amplified culture and Fig. 2 for

abcde e abcde e

FIG. 1 FIG. 2

FIG. 1. Purification of plasmid DNA (pBR322 dimer) from amplified E. coli cells. Samples were analyzed by agarose gel electrophoresis as described (4). Slots a-e contained equal aliquots (I /2000) at each stage of purification (steps 2-6, respectively) under Materials and Methods. The second slot, labeled e, was run on the same gel further away from the others to avoid possible cross-contamination and to show complete absence of RNA. Principal bands seen are: I, low-molecular-weight (transfer) RNA; 2, high-molecular-wetght (ribosomal) RNA; 3, covalently closed circular form of pBR322; 4, covalently closed circular form of pBR322 (dimer); 5, linear form of pBR322 (dimer); 6, open circular form of pBR322 (dimer); 7, region of chromosomal DNA. Other bands corresponding to higher oligomers of pBR322 are also seen.

FIG. 2. Purification of plasmid DNA (pBR322 dimer) from nonamplified E. coli cells. Details as in Fig. I.

386 MARKO, CHIPPERFIELD, AND BIRNBOIM

abcdefghi i

FIG. 3. Comparison of Hind111 sensitivity of plasmid DNA purified using glass powder (slots a-e) or phenol (slots f-j). pBR322 dimer was purified as described under Materials and Methods or by phenol/ chloroform treatment after step 5 (i.e., after proteinase K treatment). In the latter case. LiCl to 0.5 M,

CDTA to 25 mM, Tris-HCI (pH 8.0) to 25 mM, and SDS to 0.25% were added. An equal volume of phenol/chloroform (l/l) was added, and three extractions (20-min agitation at room temperature) were carried out. Residual phenol was removed by chloroform and ether extractions. SDS was removed by ethanol precipitations. Hind111 digestion conditions in both cases: 20 ~1 of 60 mM NaCI, IO mM Tris- HCI, 7 mM MgC12, 7 mM mercaptoethanol, 200 fig/ml of gelatin (pH 8.0) containing I pg of plasmid DNA and either 0, 0.5, 1, 2, or 5 units of enzyme in slots a-e and f-j, respectively. Digestion was for 1 h at 37°C. Principal bands seen are: 1, covalently closed form of pBR322; 2, linear form of pBR322; 3, covalently closed form of pBR322 (dimer); 4, linear form of pBR322 (dimer); 5, open circular form of pBR322 (dimer).

nonamplified). After the first alkali treat- ment and neutralization (a stage correspond- ing to the final step of the earlier analytical procedure (4)), some chromosomal DNA and considerable ribosomal and transfer RNA were still present (slot a in Figs. 1 and 2). After a second treatment with alkali, chromosomal DNA was diminished (slot b); after treatment with 5 M LiCl, no detectable chromosomal DNA remained and ribosomal RNA was also eliminated (slot c). Protein- ase K treatment (slot d) was used to digest residual protein and, as expected, no change in nucleic acids was seen.

The final purification step involved bind- ing to glass powder. A binding step was found to be necessary because without this additional purification, plasmid DNA was not reproducibly susceptible to digestion by restriction enzyme HindIII. The use of phenol extraction after proteinase K treat- ment did not alleviate the problem. This is

illustrated in Fig. 3. In slots a-e, 1 pg of plasmid DNA (purified on glass powder) is seen to be digested readily by increasing amounts of Hind111 enzyme. In slots f-j, 1 pg of plasmid DNA (extracted with phenol instead of binding to glass powder) was ap- preciably more resistant to equivalent amounts of Hind111 enzyme. An amount of enzyme which was sufficient to digest com- pletely the glass powder-purified DNA (slot e) caused conversion of very little phenol- treated plasmid DNA to the linear monomer (slot j). The nature of the inhibitor has not been identified but it appears not to be pro- tein because of its resistance to proteinase K and phenol extraction. After glass bind- ing, plasmid DNA was free of protein (less than 3% by weight, determined as described under Materials and Methods).

Transfer RNA was also effectively re- moved by the glass-binding procedure (Figs. 1 and 2, slot e). CDTA ( 10 mM> in LB was

found to be necessary to prevent binding of rapid method has been described for obtain- tRNA to glass; at 1 mM CDTA, some bind- ing highly purified plasmid DNA which is ing of tRNA could be seen. Under standard readily digestible by a restriction enzyme. conditions of purification, no RNA contam- We believe this method provides a useful ination was detectable (less than 3%) by the addition to the variety of techniques cur- procedure described under Materials and rently available for purifying plasmid DNA. Methods.

Glass powder has proved to be a useful Note added in proof Some additional modifications

reagent for purifying DNA (5-7). Although to the alkaline extraction procedure have recently been described (12).

plasmid DNA prepared using glass powder was chemically very pure, this step does have REFERENCES

one minor drawback. A small amount of 1. Clewell, D. B. (1972) J. Bacterial. 110, 667-676.

nicking of plasmid DNA, including what 2. Colman, A., Byers, M. J., Primrose, S. B., and

appear to be double-strand breaks, is seen Lyons, A. (1978) Eur. J. Biochem. 91,303-310.

(Figs. 1 and 2, slot e). The mechanism is 3. Vincent, W. S., III, and Goldstein, E. S. (1981)

unknown, but binding to the glass powder Anal. Biochem. 110, 123-127.

is involved since handling under similar con- 4. Birnboim, H. C., and Doly, J. (1979) Nucl. Acids

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ditions without glass powder did not cause 5. Yang, R. C.-A., Lis, J., and Wu, R. (1979) in

nicking. This amount of nicking is probably Methods in Enzymology (Grossman, L., and

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oligomers of pBR322 were detected, partic- demic Press, New York.

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Biochem. 101, 339-341.

readily recovered after glass binding. 7. Vogelstein, B., and Gillespie, D. (1979) Proc. Nat.

The yield of purified pBR322 (dimer) Acad. Sri. USA 76, 615-619.

DNA was 800 pg from 1 liter of amplified 8. Hinegardner, R. T. (1971) Anal. Biochem. 39,197-

culture and 160 pg from nonamplified cul- 201.

ture. Intensity of plasmid bands on ethidium 9. Bolivar, F., Rodriguez, R. L., Greene, P. J., Betlach,

M. C., Heyneker, H. L., Boyer, H. L., Crosa,

bromide-stained gels showed no detectable J. H., and Falkow, S. (1977) Gene 2, 85-113.

loss during purification but this is not very 10. Bock, R. M. (1967) in Methods in Enzymology

quantitative. Therefore DABA was used for (Grossman, L., and Moldave, K., eds.), Vol. 12A,

a chemical determination of DNA after pp. 224-228. Academic Press, New York.

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after the LiCl step). The overall recovery 198-200.

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ISOLATION OF PURIFIED PLASMID DNA 387

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