GWGI88
Letters in Applied Microbiology 1990, 10,209-212
An improved method for rapid isolation of plasmid DNA from wild-type Gram-negative bacteria for plasmid restriction profile analysis J.E. O L S E NDepartment of Hygiene and Microbiology and The Danish Animal Biotechnology Research Center, Royal Veterinary and Agricultural University, 13 Biilowsvej, DK-1870 Frederiksberg C., Denmark Received 20 December 1989 and accepted 21 December 1989 OLSEN, J.E. 1990. An improved method for rapid isolation of plasmid DNA from wild-type Gram-negative bacteria for plasmid restriction profile analysis. Letters in Applied Microbiology 10,209-212.
An improved method for the isolation of large and small plasmids from wild-type
Gram-negative bacteria has been developed. The protocol combines the lysis and purification procedures of two popular plasmid isolation methods, and produces DNA sufficiently pure for restriction enzyme digestion in less than three hours.
Plasmid restriction profile analysis is a convenient way of comparing plasmids from different strains of bacteria (Thompson et al. 1974; Platt et al. 1986), and has been used for the typing of wild-type plasmids; e.g. for analysis of the Salmonella typhimurium virulence-associated plasmid, pSLT (Brown et al. 1986; Platt et al. 1988). Routine use of restriction profile analysis requires a reliable and rapid method for the preparation of plasmid DNA, free from chromosomal DNA and RNA and sufficiently pure to allow enzyme digestion. Most isolation protocols, e.g. the two commonly used plasmid isolation protocols for Gram-negative bacteria described by Kado & Liu (1981) and Birmboim & Doly (1 979), use the alkaline denaturation method, introduced by Janz et al. (1966) for the preparation of replicative phages q5X 174. The Kado and Liu (1981) manual is generally the method of choice for plasmid screening, but produces DNA that is difficult to cut with restriction enzymes, and the Birmboim & Doly (1979) manual produces, in my hands, disturbing amounts of chromosomal DNA. A new protocol overcoming this problem has therefore been developed, combining lysis according to Kado & Liu (1981) with a neutralization and
purification method modified from Birmboim & Doly (1979).
Material and Methods BACTERIAL STRAINS
Strains of three different genera of Gramnegative bacteria were used; the strains and their plasmid contents are listed in Table 1. All strains were grown in LB-media [g/l: Bacto Tryptone 10; Bacto Yeast Extract 5 ; NaCl 5 (for Vibrio anguillarum 10); pH 7.41 at 37°C (V. anguillarum at 22°C). PLASMID ISOLATION
Plasmid DNA was isolated by three different methods: Kado & Liu (1981) using 0.5 ml of an overnight shaken culture; Birmboim & Doly (1979), using 1.5 ml of an overnight shaken culture [the modification by Silhavy et al. (1984), substituting potassium for sodium acetate was used]; and the new protocol developed for this study. Cells from 1.5 ml of an overnight shaken culture were harvested by centrifugation in a micro test tube and resuspended in 40 p1 50
210
J . E . Olsen Table 1. Source and plasmid contents of strains used
Strains
Plasmid contents (kb)
Salmonella ryphimurium K1466-85 dublin K228-84 dublin K660-84 Escherichia coli 39R861
94. 80. 80, 3.8* 147, 63, 36, 7
Sources B.B. Nielsen, National Veterinary Laboratory, Denmark Threlfall et al. (1986)
* Plasmid sizes estimated in this study. mmol tris, 10 mmol/l EDTA pH 8. The cells were lysed at 56°C for 30 min in 400 pl 3% SDS, 50 mmol/l tris, pH 12.45. After the addition of 300 p1 1.5 rnol/l K acetate, pH 5.2, the solution was left for 20 rnin on ice. The supernatant fluid was cleared from the precipitate by centrifugation for 10 rnin and decanted through a single layer of autoclaved surgical gauze into a clean tube holding 600 pl isopropanol, and left at room temperature for 15 min. Precipitated DNA was collected by centrifugation for 10 min. DNA was resuspended in 200 p1 10 mrnol/l Tris, 1 mmol/l EDTA pH 8.0 buffer and the sample was treated once with an equal volume
of ch1oroform:phenol (1 :1 unbuffered) [use gloves and eye protection when handling phenol and chloroform]. The phases were separated by centrifugation for 10 min and the upper phase transferred to a clean tube. One hundred p1 of 7.5 mol/l ammonium acetate and 600 pl of 96% ethanol were added and the mixture was left at 4°C for 30 min. DNA was collected by centrifugation for 6 rnin and resuspended in 100 p1 buffer. Individual digests were usually treated with RNase A (Boehringer) (1 pl of 1 mg/ml boiled RNase). All centrifugations were done in a micro centrifuge at room temperature at 15000 g.
Fig. 1. Isolation of plasmid DNA using three different plasmid isolation methods. Lane I, 111, and V: plasmids of Escherichia coli 39R861 (147, 63, 36, and 7 kb as indicated in the left legend); lane 11, IV, and VT: plasmid of Salmonella ryphimurium K1466-85 (94 kb as indicated in the right legend). Plasmids were isolated according to Kado & Liu (1981) (lanes I and II), the new protocol described in this paper (lanes 111 and IV), and Birmboim & Doly (1979)(lane V and VI). CHR, Chromosomal DNA.
Rapid isolation of plasmids
21 1
RESTRICTION ENZYME DIGESTIONS
Restriction enzyme digestions were performed for 2 h at 37°C in KGB-buffer as recommended by McClelland et al. (1988), using 12 units of HindIII or BamHI (Boehringer) per digestion. GEL ELECTROPHORESIS
Native plasmids were separated for 2.5 h in 0.8% agarose gel (Litex LSL) at a current of 100 V and restriction fragments for 3 h in 1 % agarose gels at a current of 80 V. Gels were stained in 2 mg/ml ethidium bromide (Sigma) [wear gloves when handling gels stained with ethidium bromide] and photographed under 254 nm ultraviolet light. Estimation of plasmid molecular sizes by gel electrophoresis was done as recommended by Rochelle et al. (1985); plasmids in Escherichia coli 39R861 (Threlfall et al. 1986) served as molecular size markers. Results and Discussion
As shown in Fig. 1, the method described in this paper and the Kado & ~i~ (1981) protocol, both produced plasmid DNA with Only amounts of linear (chromosomal DNA) and RNA present, as would be expected after lysis in an alkaline solution at high temperatures (Kado & Liu 1981; Kieser 1984). The surgical gauze retained small bits of precipitate after K acetateprecipitation, and also reduced the amount of chromosomal DNA (data not shown). Neutralization on ice in K or Na acetate solutions produces nicked open circular forms of plasmid DNA (Kieser 1984), and both the new and the Birmboim & Doly (1979) protocols gave rise to nicked DNA molecules (Fig. 1). Prolonged neutralization increases the proportion of nicked DNA to supercoiled DNA (Kieser 1984). DNA was routinely prepared and digested the same day, and usually plasmid-DNA for two to three digests were isolated from one preparation. As illustrated in Fig. 2, the new protocol has been used in restriction profile analysis of plasmid-DNA from Salmonella dublin. It has also been used successfully in analysis of plasmids from E . coli, and V . anguillarum (data not shown), and presumably it is adjustable to most Gram-negative bacteria. The new protocol could be scaled up for large-scale preparations including ultracentrifu-
Fig. 2. Restriction profile analysis of plasmics in Salmonella dublin using an improved plasmid isolation protocol. Lane I and 11: HindIII patterns of plasmids in Salmoneiia duhlin K660-84 and S . duhlin K228-84; lane 111: HindIII pattern of phage lambda (23.1, 9.4, 6.6, 4.4, 2.3, and 2.0 kb fragments as indicated in the legend).
gal separation of closed circular coiled molecules from linear molecules and open circular forms of DNA. Cells from 400 ml of a broth culture were collected and resuspended in 2 ml 50 mmol/l tris, 10 mmol/l EDTA, pH 8; all other reagents were scaled up proportionally. This study was supported by the Danish National Agricultural and Veterinary Research Council (grants 13-3767 and 5.23.10.01). References BIKMBOIM, H.C. & DOLY,J. 1979 A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Research 7, 15131523. BROWN,D.J., MUNRO,D.S. & PLATT, D.J. 1986 Recognition of the cryptic plasmid, pSLT, by restriction fingerprinting and a study of its incidence in Scottish Salmonella isolates. Journal of Hygiene, Cambridge 97, 193-197. JANZ, H.S., POUWELS, P.H. & SCHIPHOKST, J. 1966 Preparation of double-stranded DNA (replicative form) of bacteriophage 4x174. A simplified method. Aria Biochemica Biophysica 123,626627.
212
J . E . Olsen
KADO,C.I. & LIU, S.-T. 1981 Rapid procedure for detection and isolation of large and small plasmids. Journal of Bacteriology 145, 1365-1373. KIFSEK, T. 1984 Factors affecting the isolation of CCC DNA from Streptomyces liuidans and Escherichia coli. Plasmid 12, 19-36. MCCLELLAND, M., HANISH,J., NELSON,M. & PATEL, Y. 1988 KGB: a single buffer for all restriction endonucleases. Nucleic Acids Research 16, 364. PLATT,D.J., CHESHAM,J.S., BROWN,D.J., KKAFT, C.A. & TAGGAR?,J. 1986 Restriction enzyme fingerprinting of enterobacterial plasmids: a simple strategy with wide application. Journal of Hygiene, Cambridge 97,205-210. PLATT,D.J., TAGGART,J. & HEKAGHTY, K.A. 1988 Molecular divergence of the serotype-specific plasmid (pSLT) among strains of Salmonella typhimurium of human and veterinary origin and comparison of pSLT with the serotype-specific plasmics
of S. enferitidis and S. dublin. Journal .f Medical Microbiology 27, 217-284. ROCHELLE, P.A., FRY,J.C,, DAY, M.J. & BALE,M.J. 1985 An accurate method for estimating sizes of small and large plasmids and DNA fragments by gel electrophoresis. Journal of General Microbiology 132,53-59. SILHAVY, T.J., BEKMAN, M.L. & ENQUIST, L.W. 1984 Experiments with Gene Fusions. Cold Spring Harbor Laboratory, USA. THKELFALL, E.J., ROWE, B., FEKGUSON, L. & WARD, L.R. 1986 Characterization of plasmids conferring resistance to gentamicin and apamycin in strains of Salmonella typhirnurium phage type 204c isolated in Britain. Journal of Hygiene, Cambridge 97,419 426. THOMPSON, R., HUGHES,S.G. & BKODA,P. 1974. Plasmid identification using specific endonucleases. Molecular and General Genetics 133, 141-149.
Letters in Applied Microbiology 1990, 10,209-212
An improved method for rapid isolation of plasmid DNA from wild-type Gram-negative bacteria for plasmid restriction profile analysis J.E. O L S E NDepartment of Hygiene and Microbiology and The Danish Animal Biotechnology Research Center, Royal Veterinary and Agricultural University, 13 Biilowsvej, DK-1870 Frederiksberg C., Denmark Received 20 December 1989 and accepted 21 December 1989 OLSEN, J.E. 1990. An improved method for rapid isolation of plasmid DNA from wild-type Gram-negative bacteria for plasmid restriction profile analysis. Letters in Applied Microbiology 10,209-212.
An improved method for the isolation of large and small plasmids from wild-type
Gram-negative bacteria has been developed. The protocol combines the lysis and purification procedures of two popular plasmid isolation methods, and produces DNA sufficiently pure for restriction enzyme digestion in less than three hours.
Plasmid restriction profile analysis is a convenient way of comparing plasmids from different strains of bacteria (Thompson et al. 1974; Platt et al. 1986), and has been used for the typing of wild-type plasmids; e.g. for analysis of the Salmonella typhimurium virulence-associated plasmid, pSLT (Brown et al. 1986; Platt et al. 1988). Routine use of restriction profile analysis requires a reliable and rapid method for the preparation of plasmid DNA, free from chromosomal DNA and RNA and sufficiently pure to allow enzyme digestion. Most isolation protocols, e.g. the two commonly used plasmid isolation protocols for Gram-negative bacteria described by Kado & Liu (1981) and Birmboim & Doly (1 979), use the alkaline denaturation method, introduced by Janz et al. (1966) for the preparation of replicative phages q5X 174. The Kado and Liu (1981) manual is generally the method of choice for plasmid screening, but produces DNA that is difficult to cut with restriction enzymes, and the Birmboim & Doly (1979) manual produces, in my hands, disturbing amounts of chromosomal DNA. A new protocol overcoming this problem has therefore been developed, combining lysis according to Kado & Liu (1981) with a neutralization and
purification method modified from Birmboim & Doly (1979).
Material and Methods BACTERIAL STRAINS
Strains of three different genera of Gramnegative bacteria were used; the strains and their plasmid contents are listed in Table 1. All strains were grown in LB-media [g/l: Bacto Tryptone 10; Bacto Yeast Extract 5 ; NaCl 5 (for Vibrio anguillarum 10); pH 7.41 at 37°C (V. anguillarum at 22°C). PLASMID ISOLATION
Plasmid DNA was isolated by three different methods: Kado & Liu (1981) using 0.5 ml of an overnight shaken culture; Birmboim & Doly (1979), using 1.5 ml of an overnight shaken culture [the modification by Silhavy et al. (1984), substituting potassium for sodium acetate was used]; and the new protocol developed for this study. Cells from 1.5 ml of an overnight shaken culture were harvested by centrifugation in a micro test tube and resuspended in 40 p1 50
210
J . E . Olsen Table 1. Source and plasmid contents of strains used
Strains
Plasmid contents (kb)
Salmonella ryphimurium K1466-85 dublin K228-84 dublin K660-84 Escherichia coli 39R861
94. 80. 80, 3.8* 147, 63, 36, 7
Sources B.B. Nielsen, National Veterinary Laboratory, Denmark Threlfall et al. (1986)
* Plasmid sizes estimated in this study. mmol tris, 10 mmol/l EDTA pH 8. The cells were lysed at 56°C for 30 min in 400 pl 3% SDS, 50 mmol/l tris, pH 12.45. After the addition of 300 p1 1.5 rnol/l K acetate, pH 5.2, the solution was left for 20 rnin on ice. The supernatant fluid was cleared from the precipitate by centrifugation for 10 rnin and decanted through a single layer of autoclaved surgical gauze into a clean tube holding 600 pl isopropanol, and left at room temperature for 15 min. Precipitated DNA was collected by centrifugation for 10 min. DNA was resuspended in 200 p1 10 mrnol/l Tris, 1 mmol/l EDTA pH 8.0 buffer and the sample was treated once with an equal volume
of ch1oroform:phenol (1 :1 unbuffered) [use gloves and eye protection when handling phenol and chloroform]. The phases were separated by centrifugation for 10 min and the upper phase transferred to a clean tube. One hundred p1 of 7.5 mol/l ammonium acetate and 600 pl of 96% ethanol were added and the mixture was left at 4°C for 30 min. DNA was collected by centrifugation for 6 rnin and resuspended in 100 p1 buffer. Individual digests were usually treated with RNase A (Boehringer) (1 pl of 1 mg/ml boiled RNase). All centrifugations were done in a micro centrifuge at room temperature at 15000 g.
Fig. 1. Isolation of plasmid DNA using three different plasmid isolation methods. Lane I, 111, and V: plasmids of Escherichia coli 39R861 (147, 63, 36, and 7 kb as indicated in the left legend); lane 11, IV, and VT: plasmid of Salmonella ryphimurium K1466-85 (94 kb as indicated in the right legend). Plasmids were isolated according to Kado & Liu (1981) (lanes I and II), the new protocol described in this paper (lanes 111 and IV), and Birmboim & Doly (1979)(lane V and VI). CHR, Chromosomal DNA.
Rapid isolation of plasmids
21 1
RESTRICTION ENZYME DIGESTIONS
Restriction enzyme digestions were performed for 2 h at 37°C in KGB-buffer as recommended by McClelland et al. (1988), using 12 units of HindIII or BamHI (Boehringer) per digestion. GEL ELECTROPHORESIS
Native plasmids were separated for 2.5 h in 0.8% agarose gel (Litex LSL) at a current of 100 V and restriction fragments for 3 h in 1 % agarose gels at a current of 80 V. Gels were stained in 2 mg/ml ethidium bromide (Sigma) [wear gloves when handling gels stained with ethidium bromide] and photographed under 254 nm ultraviolet light. Estimation of plasmid molecular sizes by gel electrophoresis was done as recommended by Rochelle et al. (1985); plasmids in Escherichia coli 39R861 (Threlfall et al. 1986) served as molecular size markers. Results and Discussion
As shown in Fig. 1, the method described in this paper and the Kado & ~i~ (1981) protocol, both produced plasmid DNA with Only amounts of linear (chromosomal DNA) and RNA present, as would be expected after lysis in an alkaline solution at high temperatures (Kado & Liu 1981; Kieser 1984). The surgical gauze retained small bits of precipitate after K acetateprecipitation, and also reduced the amount of chromosomal DNA (data not shown). Neutralization on ice in K or Na acetate solutions produces nicked open circular forms of plasmid DNA (Kieser 1984), and both the new and the Birmboim & Doly (1979) protocols gave rise to nicked DNA molecules (Fig. 1). Prolonged neutralization increases the proportion of nicked DNA to supercoiled DNA (Kieser 1984). DNA was routinely prepared and digested the same day, and usually plasmid-DNA for two to three digests were isolated from one preparation. As illustrated in Fig. 2, the new protocol has been used in restriction profile analysis of plasmid-DNA from Salmonella dublin. It has also been used successfully in analysis of plasmids from E . coli, and V . anguillarum (data not shown), and presumably it is adjustable to most Gram-negative bacteria. The new protocol could be scaled up for large-scale preparations including ultracentrifu-
Fig. 2. Restriction profile analysis of plasmics in Salmonella dublin using an improved plasmid isolation protocol. Lane I and 11: HindIII patterns of plasmids in Salmoneiia duhlin K660-84 and S . duhlin K228-84; lane 111: HindIII pattern of phage lambda (23.1, 9.4, 6.6, 4.4, 2.3, and 2.0 kb fragments as indicated in the legend).
gal separation of closed circular coiled molecules from linear molecules and open circular forms of DNA. Cells from 400 ml of a broth culture were collected and resuspended in 2 ml 50 mmol/l tris, 10 mmol/l EDTA, pH 8; all other reagents were scaled up proportionally. This study was supported by the Danish National Agricultural and Veterinary Research Council (grants 13-3767 and 5.23.10.01). References BIKMBOIM, H.C. & DOLY,J. 1979 A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Research 7, 15131523. BROWN,D.J., MUNRO,D.S. & PLATT, D.J. 1986 Recognition of the cryptic plasmid, pSLT, by restriction fingerprinting and a study of its incidence in Scottish Salmonella isolates. Journal of Hygiene, Cambridge 97, 193-197. JANZ, H.S., POUWELS, P.H. & SCHIPHOKST, J. 1966 Preparation of double-stranded DNA (replicative form) of bacteriophage 4x174. A simplified method. Aria Biochemica Biophysica 123,626627.
212
J . E . Olsen
KADO,C.I. & LIU, S.-T. 1981 Rapid procedure for detection and isolation of large and small plasmids. Journal of Bacteriology 145, 1365-1373. KIFSEK, T. 1984 Factors affecting the isolation of CCC DNA from Streptomyces liuidans and Escherichia coli. Plasmid 12, 19-36. MCCLELLAND, M., HANISH,J., NELSON,M. & PATEL, Y. 1988 KGB: a single buffer for all restriction endonucleases. Nucleic Acids Research 16, 364. PLATT,D.J., CHESHAM,J.S., BROWN,D.J., KKAFT, C.A. & TAGGAR?,J. 1986 Restriction enzyme fingerprinting of enterobacterial plasmids: a simple strategy with wide application. Journal of Hygiene, Cambridge 97,205-210. PLATT,D.J., TAGGART,J. & HEKAGHTY, K.A. 1988 Molecular divergence of the serotype-specific plasmid (pSLT) among strains of Salmonella typhimurium of human and veterinary origin and comparison of pSLT with the serotype-specific plasmics
of S. enferitidis and S. dublin. Journal .f Medical Microbiology 27, 217-284. ROCHELLE, P.A., FRY,J.C,, DAY, M.J. & BALE,M.J. 1985 An accurate method for estimating sizes of small and large plasmids and DNA fragments by gel electrophoresis. Journal of General Microbiology 132,53-59. SILHAVY, T.J., BEKMAN, M.L. & ENQUIST, L.W. 1984 Experiments with Gene Fusions. Cold Spring Harbor Laboratory, USA. THKELFALL, E.J., ROWE, B., FEKGUSON, L. & WARD, L.R. 1986 Characterization of plasmids conferring resistance to gentamicin and apamycin in strains of Salmonella typhirnurium phage type 204c isolated in Britain. Journal of Hygiene, Cambridge 97,419 426. THOMPSON, R., HUGHES,S.G. & BKODA,P. 1974. Plasmid identification using specific endonucleases. Molecular and General Genetics 133, 141-149.
