Biomedical Engineering Reference
In-Depth Information
restriction is not involved. Groups of plasmids which
are mutually incompatible are considered to belong
to the same incompatibility (Inc) group. Over 30 in-
compatibility groups have been defined in
E. coli
and
13 for plasmids of
S. aureus.
Plasmids will be incom-
patible if they have the same mechanism of replica-
tion control. Not surprisingly, by changing the
sequence of the RNA I/RNA II region of plasmids
with antisense control of copy number, it is possible
to change their incompatibility group. Alternatively,
they will be incompatible if they share the same
par
region (Austin & Nordstrom 1990, Firsheim & Kim
1997).
Upper band containing
chromosomal DNA and
open plasmid circles
The purification of plasmid DNA
An obvious prerequisite for cloning in plasmids is
the purification of the plasmid DNA. Although a
wide range of plasmid DNAs are now routinely
purified, the methods used are not without their
problems. Undoubtedly the trickiest stage is the lysis
of the host cells; both incomplete lysis and total
dissolution of the cells result in greatly reduced
recoveries of plasmid DNA. The ideal situation
occurs when each cell is just sufficiently broken to
permit the plasmid DNA to escape without too much
contaminating chromosomal DNA. Provided the
lysis is done gently, most of the chromosomal DNA
released will be of high molecular weight and can be
removed, along with cell debris, by high-speed
centrifugation to yield a
cleared lysate
. The produc-
tion of satisfactory cleared lysates from bacteria
other than
E. coli
, particularly if large plasmids are to
be isolated, is frequently a combination of skill, luck
and patience.
Many methods are available for isolating pure
plasmid DNA from cleared lysates but only two will
be described here. The first of these is the 'classical'
method and is due to Vinograd (Radloff
et al.
1967).
This method involves isopycnic centrifugation of
cleared lysates in a solution of CsCl containing
ethidium bromide (EtBr). EtBr binds by intercalating
between the DNA base pairs, and in so doing causes
the DNA to unwind. A CCC DNA molecule, such as
a plasmid, has no free ends and can only unwind to
a limited extent, thus limiting the amount of EtBr
bound. A linear DNA molecule, such as fragmented
chromosomal DNA, has no such topological con-
Lower band of
covalently closed
circular plasmid DNA
Fig. 4.6
Purification of Col E1
Kan
R
plasmid DNA by
isopycnic centrifugation in a CsCl-EtBr gradient.
(Photograph by courtesy of Dr G. Birnie.)
straints and can therefore bind more of the EtBr
molecules. Because the density of the DNA-EtBr
complex decreases as more EtBr is bound, and
because more EtBr can be bound to a linear molecule
than to a covalent circle, the covalent circle has a
higher density at saturating concentrations of EtBr.
Thus covalent circles (i.e. plasmids) can be separated
from linear chromosomal DNA (Fig. 4.6).
Currently the most popular method of extracting
and purifying plasmid DNA is that of Birnboim and
Doly (1979). This method makes use of the observa-
tion that there is a narrow range of pH (12.0 -12.5)
within which denaturation of linear DNA, but not
covalently closed circular DNA, occurs. Plasmid-
containing cells are treated with lysozyme to weaken
the cell wall and then lysed with sodium hydroxide
and sodium dodecyl sulphate (SDS). Chromosomal
DNA remains in a high-molecular-weight form but
is denatured. Upon neutralization with acidic sodium
acetate, the chromosomal DNA renatures and aggreg-
ates to form an insoluble network. Simultaneously,
