Letters in Applied Microbiology 1992, 14, 250-254

An improved method for rapid purification of covalently closed circular plasmid DNA over a wide size range A . K . A Z A D ,J . G . COOTE*& R . P A R T O NDepartment of Microbiology, University of Glasgow, Glasgow, U K D S T / 4 : received 13 January 1992 and accepted 14 February 1992

A Z A D , A . K . , COOTE,J.G. & P A R T O NR, . 1992. An improved method for rapid

purification of covalently closed circular plasmid DNA over a wide size range. Letters in Applied Microbiology 14, 250-254. An improved method has been developed for the large-scale purification of covalently closed circular (CCC) plasmid DNA molecules of sizes ranging from 4.3 to 73 kb. This protocol uses an alkaline-lysis procedure followed by acid-phenol extraction but with several modifications to previously reported methods. The principal modification is the replacement of NaCl by MgCI, in the extraction buffer to improve yield and to remove chromosomal and other non-CCC plasmid DNA. Plasmid DNA can be purified in less than 1 h and used successfully in restriction enzyme analysis and cloning experiments.

Restriction analysis and further characterization of plasmids often require extraction of purified plasmid DNA on a large scale. In addition, the identification and detection of numbers of different plasmids present in a bacterium involve the purification of CCC plasmid DNA. The method of Birnboim & Doly (1979), based on alkaline lysis of bacteria, can be used on a large scale to produce a good yield of plasmid DNA but the preparation is frequently contaminated with chromosomal DNA and nicked open circular (OC) forms of the plasmid (Kieser 1984; Olsen 1990). These authors reported improved methods for the small-scale preparation of plasmid DNA free from chromosomal DNA and suitable for restriction analysis. However, both methods use alkaline denaturation in the presence of sodium dodecyl sulphate at high temperatures (56"-70"C). This is not suitable for the large-scale preparation of plasmid DNA due to the high viscosity of the lysed bacterial pellet and the fact that certain strains of Escherichia coli, such as HB101, release large amounts of carbohydrate during heating and this severely interferes with the final purification process

* Corresponding author.

(Sambrook et a/. 1989). The method of choice widely used for the large-scale purification of CCC DNA is caesium chloride-ethidium bromide density-gradient centrifugation (Radloff et a/. 1967) which is expensive and timeconsuming. Zasloff et d. (1978) developed a large-scale purification procedure for CCC plasmid DNA which avoided prolonged centrifugation and used acidified phenol at low ionic strength. This procedure selectively extracts chromosomal and non-CCC plasmid DNA into the phenolic phase leaving CCC forms in the aqueous phase. However, this method was applied to plasmids of limited size range. Here we report an improved and rapid procedure, based on that of Zasloff et al. (1978), for the large-scale purification of CCC plasmid DNA over a wide size range. This new protocol combines a modified alkaline-denaturation procedure with a modified version of the acidphenol extraction. Materials and Methods BACTERIAL STRAINS A N D PLASMIDS

Four E . coli strains were used as the hosts for five plasmid species of different sizes (Table 1).

Rapid purtfication of plasmid DNA

251

Table 1. Strains and Dlasmids used Strain o r plasmid Eschrrichia co/r JM83 JC3272 HBlOl

J 53 Plasmids pPH843 pRM3022 (original R,,,) pRK2013 RP4

Source or reference

ara A (lac-proAB) rpaL cpbOdlacZAMl5 His-Lys-Trp -SmR supE44 hsdS20 ( r e m i ) recA13 ara-14 proA2 lacy1 g a l K 2 rpsl20 xyl-5 mtl-1 Pro- Met-

Vieira 6i Messing (1982) Achtman et a/. (1971) Sambrok et ul. (1989)

4.3 kb, ApR 4.4 kb, ApR

H.A. Gibbs, Glasgow University, UK

48 kb, KmR, IncP, T r a + , ColEl 54 kb, Ap", KmR, TcR,IncP, Tra' 73 kb, pRK2013 kan::Tn7 xyz::TnS KmR.TD". SD". SmR

pUW964 ~

Relevant characteristics*

~

~

D.J. Platt, Glasgou Royal Infirniary. UK

E.R. Moxon, Oxford University. UK Figurski & Helinski (1979) D.J. Platt, Glasgow Royal Infirmary, UK A.A. Weiss, Virginia University, USA

~~

*

His, Histidine; Lys, lysine; Trp, tryptophan; Pro, proline; Met, methionine; kb, kilobases; ', resistance; Sm, streptomycin; Ap, ampicillin; Kmlkan, kanamycin; Tc, tetracycline; Tp, trimethoprim; Sp, spectinomycin; Inc, incompatibility group; T r a + , transfer gene functions; ColE I. colicin E 1-1ype replicon; Tn, transposon

PLASMID ISOLATION

Plasmids pPH843 and pRM3022 were amplified in E. coli JM83 by growing the organism in Terrific broth (g/l: tryptone, 12; yeast extract, 24; glycerol, 4 ml; KH,PO,, 2.3 1; K,HPO, , 12.54) (Tartof & Hobbs 1987) supplemented with 1% (w/v) yeast nitrogen base (Difco, Detroit, MI) and chloramphenicol (Sigma, St Louis, MO) (170 pg/ml) as described previously (Azad et al. 1992). Escherichia coli K-12 strain 553 contaiing plasmid RP4, strain HBlOl containing plasmid pUW964 and a JC3272 transconjugant strain containing both pRK2013 and pPH843 were grown in Brain Heart Infusion Broth (Oxoid, Basingstoke, UK). Plasmid DNA was isolated from 500 ml cultures, shaken overnight at 3 7 T , according to the modified alkaline-lysis method of Birnboim & Doly (1979) as described by Sambrook ef al. (1989). However, an additional step included during the extraction procedure was the precipitation of plasmid DNA with an equal volume of polyethylene glycolNaCl solution (PEG 8000; Sigma, 13%; NaC1, 1.6 mol/l) to remove small oligonucleotides immediately before the phenol-chloroform extraction step. Plasmid DNA preparation was taken up in 10 ml of low ionic strength T E buffer (Tris 10 mmol/l; sodium-EDTA, 1 mmol/l; ph 8.0), extracted with an equal

volume of 100 mmol/l Tris.HC1 (pH 8.0)saturated phenol and chloroform (1 : I), reextracted with 10 ml of chloroform mixture (chloroform : isoamyl alcohol; 24 : 1) and 10 ml of 5 mol/l ammonium acetate was then added to precipitate chromosomal DNA. Isopropanol(40 ml) was used at room temperature to precipitate plasmid DNA which contained RNA, some chromosomal DNA and different topological forms of plasmid DNA. The preparation was taken up in 500 pl of TE buffer (pH 8.0) and treated with 20 pg/ml ribonuclease A (Sigma) at 37'C for 30 min to remove RNA.

CCC PLASMID PURIFICATION

To a 1.5 ml microcentrifuge tube containing a maximum of 300 pl of plasmid DNA (50-70 pg/ml) in T E buffer (pH 8.0) was added 30 pl of 500 mmol/l sodium acetate (pH 4.0) and 33 p1 of 15 mmol/l MgCI, (AnalaRR; BDH, Poole, UK) instead of 750 mmol/l NaCI, as used by Zasloff et a[. (1978). An equal volume of phenol (Formachem, Strathhaven, UK) equilibrated with 50 mmol/l sodium acetate (pH 4.0) (1 : I ; w/v) was added and the mixture vortexed vigorously for 1 min. The mixture was centrifuged at 15OOO g in a microcentrifuge (Biofuge A, Heraeus Sepatech, Germany) for 5 min and the

252

A . K . Azad et al.

supernate was removed to a fresh tube and neutralized by adding 36 p1 of 500 mmol/l Tris.HC1 (pH 8.6) and mixed by gentle agitation. Then 396 p1 of chloroform mixture were added and, after brief vortexing, the mixture was centrifuged at 15000 g for 3 min. The supernate was removed to a fresh tube to which 39 p1 of 3 mol/l sodium acetate (pH 6.0) and 860 pl of ethanol were added and, after mixing by gentle inversion, the tubes were left at room temperature for 10 min. The purified CCC plasmid DNA was recovered by centrifugation at 15000 g for 10 min, washed with 70% ethanol and resuspended in TE buffer or distilled H,O. All manipulations in the purification procedure were carried out at room temperature.

GEL ELECTROPHORESIS

Plasmids were separated on either a 0.7% or a

0.8% (w/v) agarose (type 11-A, Sigma) gel in TBE buffer (Tris, 89 mmol/l: boric acid, 89 mmol/l; sodium-EDTA, 2 mmol/l; pH 8.0) at a constant current of 60 mA for 4 h using a horizontal electrophoresis apparatus (GNA-200; Pharmacia, Uppsala, Sweden). Gels were stained in 1 pg/ml ethidium bromide (Sigma) solution for 20 min and photographed under ultraviolet light with a Polaroid MP-4 Land camera. A supercoiled DNA ladder (GibcoBRL, Paisley, UK) and HindIII-digested 1. DNA (Sigma) were used as molecular size standards. When NindIII-digested A. DNA fragments were used as size markers, the supercoiled equivalent plasmid DNA size was determined by the method of Platt & Taggart (1987).

Results and Discussion Several modifications were made to the original protocol of Zasloff et al. (1978). These included the use of: (i) MgCI, instead of NaCl in the low ionic strength buffer; (ii) undistilled phenol instead of distilled phenol; (iii) vortexing for 1 min instead of shaking for 2-3 min; (iv) only one acid-phenol extraction instead of up to four extractions; (v) a single extraction with chloroform-isoamyl alchol instead of four extractions with anhydrous ether to remove phenol; and (vi) room temperature instead of 04°C. Furthermore, in the modified protocol, purified CCC DNA was finally precipitated in

300 mmol/l sodium acetate (pH 6.0) and ethanol. Several different concentrations (1-50 mmol/l) of MgCI, were used to replace NaCI, but 1.5 mmol/l MgC1, was found most effective in CCC DNA purification (data not shown). Figure 1 illustrates the preparation of plasmid DNA by the alkaline-lysis method (lane 1, each group) which was then purified by acid-phenol extraction (lanes 2 and 3, each group). The difference between purification of CCC plasmids with 75 mmol/l NaCl (lane 2, each group) and with 1.5 mmol/l MgCI, (lane 3, each group) in the low ionic strength buffer is clearly seen. The use of NaCl at the optimum concentration (75 mmol/l) specified by Zasloff et a!. (1978) produced a diffused DNA smear in each case and removed CCC plasmid DNA molecules of higher molecular weight either partially (lane 2, group C) or completely (lane 2, groups D and E). This suggested that NaCl might allow degradation of high molecular weight DNAs to small linear fragments, which were not efficiently extracted into phenol. This is in agreement with Zasloff et a!. (1978) who reported that small oligoribonucleotides and linear DNA fragments of less than 1.5 kb could not be removed by their procedure. The physical basis of acid-phenol extraction of CCC DNA molecules and Mg2+-DNA interactions were described by Miiller et al. (1983). Both phenol and acid pH at low ionic strength denature non-CCC DNA molecules by converting them into single-stranded linear forms which are extracted from the water phase into the phenolic phase due to their hydrophobic nature and charge compensation by Mg2+ ions. Moreover, it is likely that divalent Mgz+ ions bind to and interact with denatured DNA, which has exposed phosphate groups, more than monovalent Na+ ions; Mgz+ ions were reported to have a high affinity for binding to DNA (Sander & Ts'o 1971). At present, we do not have any explanation for the loss of CCC plasmids of higher molecular weight (lane 2, groups C-E) in the presence of Na+ ions. Vortexing was a crucial step in obtaining a good yield of CCC molecules, because more than one extraction with acid-phenol, as indicated by Zasloff et al. (1978), in the presence of NaCl resulted in further loss of CCC DNA. However, a single extraction with MgCI, in place of NaCl was sufficient for complete purification of CCC plasmid DNA forms. Crude plasmid DNA preparations

Rapid purification of plasmid D N A A

1

1

2

3

D

C

B

1

253 c

r I

7

3

kb

kb

10.1 8.1 7

23-I

6 5

9.4

6.6

4

4.4

3 2. I

2.3 2.0

Fig. I Agarose gel (0.8”.,for groups A and B. 0.7”,, for groups C-E) electrophoresis o f plasmid DNA preparations extracted on a large scale h) the alkaline-lysis method (lane I . each group) and then purified by acidphenol extraction using either 75 mmol 1 NaCl (Zasloff er a / . 1978) (lane 2. each group) or 1.5 mmol 1 MgClz (new protocol) (lane 3. each group) in the 10% ionic strength buffer. Five to seven microlitres from a total amount of 500 pl of crude or purified plasmid DNA samples derived from one large-scale preparation were run in each lane. Group A: plasmid pPH843 (4.3 hb); group B . plasmid pRM3022 (4.4 kb); g w u p C . plasmids pRK2013 (48 kb) and pPH843 (4.3 k b ) : group D : plasmid RP4 (54 kb): group E: plasmid pUW964 (73 kh). The faint upper bands in lane 3 of group B probabl) represent a ‘step ladder’ of 1,4-kh CCC molecules with different numbers of supercoil turns (Kieser 1984). A supercoiled DNA ladder (lane I ) ,ind HindIII-digested I DNA (lane 11) were used as molecular si7e marker$

at both high (lane 1. groups A and B) and low (lane I , groupa C-E) concentrations, without adjustment to 10 A,,, unitsiml as suggested by Zasloff er a/. (1978), could be purified equally well by the present protocol. A plasmid of 73 kb (lane 3, group E) was readily purified by this procedure. whereas a plasmid of 38 kb was the largest purified by Zasloff et ul. (1978). This combined protocol was also applied to another Gram-negative bacterium. Pasreirrellu haemolytica, and found to be effective for the purification of plasmid DNA ranging from 4.3 kb to 20 kb (data not shown). The procedure i5 simple a n d rapid: crude plasmid DNA preparations can he purified in less than h. For the preparation Of ccc plasmid DNA i t therefore offers a ready alternative either to caesium chloride-ethidium bromide denslt).-gradient centrifugation, which is time-consuming. o r to commercially available purification kits, which are expensive.

We thank Dr I). on this work.

J.

for

comments

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SANDER, C. & Ts'o, P.O.P. 1971 Interaction of nucleic acids. VIII. Binding of magnesium ions by nucleic acids. Journal ofMolecular Biology 55, 1-21. TARTOF,K.D. & HOBBS,C.A. 1987 Improved media for growing plasmid and cosmid clones. Bethesda Research Laboratory Focus 9, 12. VIEIRA,J. & MESSING, J. 1982 The pUC plasmids, an M 13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 19,259-268. ZASLOFF,M., GINDER,G.D. & FELSENFELD, G. 1978 A new method for the purification and identification of covalently closed circular DNA molecules. Nucleic Acids Research 5, 1139-1151.