Download A Rapid Method for the Identification of Plasmid Desoxyribonucleic

PLASMID
1,
584-588 (1978)
A Rapid Method for the Identification
of Plasmid
Desoxyribonucleic
Acid in Bacteria
THOMASECKHARDT
Department
of Microbiology,
New York University Srhool of Medicine,
New York, New York 10016
Accepted June 14, 1978
A fast and very sensitive procedure is described for detecting plasmids in bacterial strains.
The size of plasmids is determined by agarose gel electrophoresis.
Plasmids present in one
or more copies per cell with a molecular mass ranging from 2 to over 150 megadaltons
may be identified.
Currently two types of rapid screening
techniques for plasmid desoxyribonucleic
acid (DNA) are used (1,4,5,7). One type requires little starting material, but subjects
the DNA to considerable stress during lysis
(5,7) or during separation of plasmid DNA
from chromosomal DNA (1) and is therefore
not suitable for large plasmids [greater than
50 megadaltons (Md)] or small plasmids with
a low copy number. The other type (4) is
time consuming, requiring several steps for
plasmid isolation and separation from the
chromosomal DNA.
The aim in the design of the technique
presented here was to combine the advantages of the above-mentioned techniques
and to avoid the disadvantages inherent in
them. A gentle lysis procedure and minimization of the manipulations of DNA lysate
were used to develop a very sensitive technique with a good yield of circular covalently
closed (CCC) plasmid DNA. The bacteria
(between lo7 and lo* cells from a liquid culture or one to two single colonies) are lysed
directly in the slots of an agarose gel. The
chromosomal and plasmid DNA are then
separated by electrophoresis. Since the bulk
of the chromosomal DNA is intact, it is excluded from the gel under the conditions
used; usually less than 0.5% of the total
chromosomal DNA migrates into the gel as a
band of linear DNA. Plasmids with a mole0147-619X/78/0014-0584$02.00/0
CopYridS
0 1978 by Academic Press, Inc.
All rights of reproduction
in any form reserved.
584
cular mass ranging from 2 to over 150 Md
and present in one or more copies per cell
can be visualized within 3 to 4 h. The procedure has been carried out with a variety
of bacterial species including Bacillus
subtilis, Escherichia
coli, Pseudomonas
aeruginosa,
Pseudomonas
phaseolicola,
Pseudomonas pseudoalcaligenes, and Neisseria ghonorrhea.
MATERIALS
AND METHODS
The conditions used to lyse gram-negative
bacteria are slightly different from those for
gram-positive bacteria (B. subtilis) and are
therefore described separately.
Lysozyme mixture for gram-negative bacteria . The lysozyme mixture for gram-nega-
tive bacteria was composed of lysozyme
(Calbiochem, 7500 U/ml), ribonuclease I
(Worthington, 0.3 U/ml), and 0.05% bromphenol blue in Tris-borate buffer (pH 8.2;
89 mM Tris base, 12.5 mM disodium EDTA,
and 8.9 mM boric acid), and 20% Ficoll
400,000 (Sigma). The ribonuclease is first
dissolved in 0.4 M sodium acetate buffer,
pH 4.0, at 10 mg/ml and heated for 2 min
at 98°C before diluting it into the rest of
the lysozyme mixture.
Lysozyme mixture for gram-positive bacteria. This mixture was similar to the one
for gram-negative bacteria but contained
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75,000 U/ml lysozyme, 50 mM disodium
EDTA (pH 8.0), and 0.1 M sodium chloride.
Both lysozyme mixtures are stable for several months at room temperature.
SDS mixture for gram-negative
bacteria.
This mixture was composed of 0.2% sodium
dodecyl sulfate (SDS) in Tris-borate buffer
(89 mM Tris base, 2.5 mM disodium EDTA,
and 8.9 mM boric acid) in 10% Ficoll400,OOO.
SDS mixture for gram-positive
bacteria.
This mixture was similar to the one for gramnegative bacteria but contained 2% SDS.
Overlay mixture for gram-negative
and
gram-positive
bacteria. The overlay mix-
ture was the same as the SDS mixture for
gram-negative bacteria except it contained
5% Ficoll 400,000.
Electrophoresis
procedure. For electrophoresis a standard vertical slab gel apparatus (gel dimensions: 110 x 140 x 2.5 mm)
with 12 slots (6.5 x 15 x 2.5 mm) is used.
Gel concentrations vary from 0.75 to 1.2%
Agarose (Seakem, type ME, Marine Colloids, Inc.) in electrophoresis buffer (89 mM
Tris base, 2.5 mM disodium EDTA, and 8.9
mM boric acid).
Loading and lysis procedure. Either single
colonies from plates (24 to 72 h old) or liquid
cultures are used as sources of plasmid
DNA. The procedure involves the following steps.
(i) One or two single colonies (10’ to IO8
cells) are picked with the flat end of a toothpick and resuspended in 15~1 of the lysozyme
mixture which had been previously put into
the empty slots of the mounted gel. The
suspension becomes slightly turbid. If cells
from a liquid culture are used, a suitable
volume (0.1 to 0.5 ml) is centrifuged and
resuspended in 10~1of electrophoresis buffer
with 20% Ficoll 400,000 and the cell suspension is mixed with the lysozyme mixture in the gel slot. Gram-negative bacteria
are left for 2 to 5 min at room temperature,
and gram-positive bacteria are left for 30 to
45 min.
(ii) Thirty microliters of the SDS mixture
is carefully layered on top of the bacteria-
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lysozyme mixture and the two layers are
gently mixed with a toothpick, moving it
from side to side (not more than twice for
gram-negative bacteria). Complete mixing
should be avoided and the two layers should
still be distinguishable.
(iii) One hundred microliters of the overlay mixture is layered on top of the SDSlysozyme layers without disturbing the now
viscous DNA lysate.
(iv) The slots are sealed with hot agarose
(SO’C) and both chambers are filled with
electrophoresis buffer. The plasmid DNA is
electrophoresed for 60 min at 2 mA and then
for 60 to 150 min at 40 mA (depending on
the plasmid size and the separation desired). The gel is stained with ethidium bromide (0.4 pg/ml) in electrophoresis buffer
for 15 min and then photographed under uv
light using a short-wave transilluminator
(type C61 from Ultraviolet Products, Inc.)
and Polaroid type 55 or 57 film and a red
filter (Wratten number 25).
Notes on the procedure. It is important
that in Step (i) that the gel is not overloaded
with cells, since this reduces the yield of
intact plasmid DNA and simultaneously increases the chromosomal background drastically. Different amounts of liquid culture
should be used in initial trials to get an idea
of the optimal amount of cells required. For
the lysis of B. subtilis it is important to use
vegetative cells. Therefore an exponentially
growing culture was used routinely. The
cells were resuspended directly in the
lysozyme mixture and the whole mixture
was transferred into an empty slot of the
gel. This is feasible since B. subtifis cells
are not lysed immediately by the lysozyme
mixture, in contrast to the gram-negative
bacteria. Incomplete mixing in Step (ii) was
found to be necessary in all cases to obtain
an optimal yield of plasmid DNA. Presumably partial lysis occurs at this stage
and lysis is completed by migration of the
sodium dodecyl sulfate through the bacteria-lysozyme layer during electrophoresis.
Adding the overlay mixture in Step (iii) al-
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FIG. 1. Agarose gel, 0.78% CCC plasmid DNA (O), OC plasmid DNA (X); the position of chromosomal
DNA is indicated by an arrow at the left. (1) pE194cop6; (2) and (3) pE194, 10” and 2 x 10’ cells used, respectively; (4) pSClO1; (5) pBR322; (6) pEC125; (7) pEC124 and F’lac; (8) R 1; and (9) F’ser. For a detailed
description of the strains see the text. Electrophoresis conditions: 1 h at 2 mA, followed by 2 h at 40 mA. Migration of the chromosomal band was 5.9 cm from the top (anode); the bromphehol blue marker migrated out
of the gel.
lows the stirred up DNA to settle again and
avoids smearing of DNA in the gel.
RESULTS AND DISCUSSION
Typical gel patterns of plasmids of various
sizes are shown in Figs. 1 and 2. Figure 1
shows the migration pattern in a 0.78%
agarosegel, and Fig. 2 shows the same strains
in a 1% agarose gel. The two different gel
concentrations are used to distinguish
whether a given DNA band is formed either
by linear DNA or by open circular (OC) or
CCC plasmid DNA, since the higher gel concentration retards the migration of OC and
CCC plasmid DNA more than that of the
linear (e.g. chromosomal) DNA (5). CCC
DNA is indicated on the left side of the slot
by 0 , and OC DNA by X. The position
of the chromosomal linear DNA in the range
of 15 to 20 Md is indicated on the left side
of the gel by an arrow. The size and nature
of this DNA were verified by using nonplasmid-bearing strains and linear molecular
mass standards. Slots 1 to 3 show B. subtifis
plasmids, and slots 4 to 9 illustrate plasmids
in E. coli. Slot 1 shows a copy number
mutant of plasmid pE194, pE194cop6 (2 Md),
with the starting material being IO7 cells.
The CCC plasmid DNA migrates at both
gel concentrations in front of the chromosomal DNA band. Between the CCC DNA
and the chromosomal band two or three intermediate bands are visible. It is not known
whether they represent OC DNA or multimers of pE 194~0~6. Slots 2 and 3 show the
parental plasmid pE194, present in only one
copy per cell (L. Mindich, personal communication). In slot 2 lo* cells were used,
and in slot 3, 2 x 10’ cells were used. Even
at the lower cell concentration enough plas-
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FIG. 2. Agarose gel, 1%. For explanation of the symbols see the legend to Fig. 1. The electrophoresis c:onditions were the same as those given in the legend in Fig. 1. Migration of the chromosomal band was 4.7
cm from the top.
mid DNA is released to form a visible band.
The patterns of the bands in slots 1,2, and 3
are similar. Slot 4 shows plasmid pSC 101,
a 5.6-Md plasmid with a low copy number
of about 7 (3). At the lower gel concentration the CCC plasmid DNA migrates in front
of the chromosomal DNA, and at the higher
gel concentration the CCC DNA migrates
behind it. Slot 5 shows pBR322, a plasmid
of 2.6 Md with high copy number (over 20)
[see Ref. (2)] which is frequently used as a
cloning vehicle for recombinant DNA. Slot
6 shows pEC125, a recombinant plasmid
with a molecular mass of 7.1 Md derived
from pBR322. At the lower gel concentration (Fig. 1) the CCC plasmid DNA comigrates with the chromosomal band; at 1%
agarose concentration the CCC plasmid
DNA moves behind the band. Slot 7 shows
the plasmids F’lac [96 Md, see Ref. (6)] and
pEC124 (4 Md) present in the same strain.
pEC124 is a recombinant plasmid derived
from pBR322. The multiple CCC DNA bands
of pEC124 observed at the higher get concentration are an artifact due to the high
plasmid concentration at this agarose concentration; they are not found at the lower
concentration. A comparison of the migration rate at the two gel concentrations shows
that the smaller molecule is relatively more
retarded at the higher gel concentration than
the larger F’lac. Slot 8 shows plasmid R 1
[61 Md, see Ref. (2)]. The band moving in
front of the chromosomal DNA has the same
relative migration rate relative to the
chromosomal DNA at both gel concentrations and is therefore not CCC or OC plasmid DNA. Bands of this type are characteristic for some of the larger plasmids
(see slot 9) and they disappear if the plasmid
is absent from the host strain. The origin of
this band is not known. Slot 9 shows a F’ser
plasmid (over 140 Md). It gives rise to segregant plasmids of smaller size, as indicated
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by the two fainter bands appearing below the
CCC plasmid DNA.
The technique described here is useful for
many purposes because of its sensitivity,
simplicity, and the variety of bacterial
species it can be applied to. The migration
rate was found to be inversely related to the
logarithm of the plasmid mass in the 2- to
50-Md range in a 0.8% agarose gel as
noted elsewhere (4) (data not shown). The
technique has been used to identify plasmids
present in a given strain, to screen mixtures
of recombinant plasmids for those of a desired size, and to investigate the fate of unstable plasmids in different host backgrounds.
ACKNOWLEDGMENTS
I am very grateful to Dr. Werner Maas for support
and helpful discussion of the manuscript and to Dr.
L. Mindich and Dr. D. Dubnau for providing some
of the strains and data prior to publication. This work
was supported by USPHS Grant 5RO GM 06048 to
W. Maas.
REFERENCES
I. BARNES, W. M., Science 195, 393-395 (1977).
2. BUKHARI, A. I., SHAPIRO, J. A., AND ADHYA,
S. L., “DNA Insertion Elements, Plasmids, and
Episomes,” Appendix B, pp 607-670. Cold
Spring Harbor Laboratory, Cold Spring Harbor,
N. Y., 1977.
3. CABELLO, F., TIMMIS, K., AND COHEN, S. N.,
Nature (London) 259, 285-290 (1976).
4. MEYERS, T. A., SANCHEZ, D., ELWELL, L. P.,
AND FALKOW, S., .I. Bacterial.
127, 15291537 (1976).
5. MICKEL, S., ARENA, V., AND BAUER, W., Nucleic
Acid Res. 4, 1465-1482 (1977).
6. PALCHAUDHURI, S., AND MAAS, W. K. Mol. Gen.
Genet. 146, 215-231 (1976).
7. TELFORD, T., POSELEY, P., SCHAFFNER, W., AND
BIRNSTIEL, M., Science 195, 391-392 (1977).