Biomedical Engineering Reference
In-Depth Information
the high concentration of sodium acetate causes
precipitation of protein-SDS complexes and of high-
molecular-weight RNA. Provided the pH of the
alkaline denaturation step has been carefully con-
trolled, the CCC plasmid DNA molecules will remain
in a native state and in solution, while the contamin-
ating macromolecules co-precipitate. The precipitate
can be removed by centrifugation and the plasmid
concentrated by ethanol precipitation. If necessary,
the plasmid DNA can be purified further by gel
filtration.
Recently a number of commercial suppliers of con-
venience molecular-biology products have developed
kits to improve the yield and purity of plasmid DNA.
All of them take advantage of the benefits of alkaline
lysis and have as their starting-point the cleared
lysate. The plasmid DNA is selectively bound to an
ion-exchange material, prepacked in columns or
tubes, in the presence of a chaotropic agent (e.g.
guanidinium hydrochloride). After washing away
the contaminants, the purified plasmid is eluted in a
small volume.
The yield of plasmid is affected by a number of
factors. The first of these is the actual copy num-
ber inside the cells at the time of harvest. The copy-
number control systems described earlier are not the
only factors affecting yield. The copy number is also
affected by the growth medium, the stage of growth
and the genotype of the host cell (Nugent
et al.
1983,
Seelke
et al.
1987, Duttweiler & Gross 1998). The
second and most important factor is the care taken
in making the cleared lysate. Unfortunately, the
commercially available kits have not removed the
vagaries of this procedure. Finally, the presence in
the host cell of a wild-type
end
A gene can affect the
recovery of plasmid. The product of the
end
A gene
is endonuclease I, a periplasmic protein whose
substrate is double-stranded DNA. The function
of endonuclease I is not fully understood. Strains
bearing
end
A mutations have no obvious phenotype
other than improved stability and yield of plasmid
obtained from them.
Although most cloning vehicles are of low molecu-
lar weight (see next section), it is sometimes neces-
sary to use the much larger conjugative plasmids.
Although these high-molecular-weight plasmids
can be isolated by the methods just described, the
yields are often very low. Either there is inefficient
release of the plasmids from the cells as a conse-
quence of their size or there is physical destruction
caused by shear forces during the various manipulat-
ive steps. A number of alternative procedures have
been described (Gowland & Hardmann 1986), many
of which are a variation on that of Eckhardt (1978).
Bacteria are suspended in a mixture of Ficoll and
lysozyme and this results in a weakening of the cell
walls. The samples are then placed in the slots of an
agarose gel, where the cells are lysed by the addition
of detergent. The plasmids are subsequently extracted
from the gel following electrophoresis. The use of
agarose, which melts at low temperature, facilitates
extraction of the plasmid from the gel.
Desirable properties of
plasmid cloning vehicles
An ideal cloning vehicle would have the following
three properties:
• low molecular weight;
• ability to confer readily selectable phenotypic traits
on host cells;
• single sites for a large number of restriction
endonucleases, preferably in genes with a readily
scorable phenotype.
The advantages of a low molecular weight are
several. First, the plasmid is much easier to handle,
i.e. it is more resistant to damage by shearing, and
is readily isolated from host cells. Secondly, low-
molecular-weight plasmids are usually present as
multiple copies (see Table 4.2), and this not only
facilitates their isolation but leads to gene dosage
effects for all cloned genes. Finally, with a low
molecular weight there is less chance that the vector
will have multiple substrate sites for any restriction
endonuclease (see below).
After a piece of foreign DNA is inserted into a
vector, the resulting chimeric molecules have to
be transformed into a suitable recipient. Since the
efficiency of transformation is so low, it is essential
that the chimeras have some readily scorable
phenotype. Usually this results from some gene, e.g.
antibiotic resistance, carried on the vector, but could
also be produced by a gene carried on the inserted
DNA.
One of the first steps in cloning is to cut the vector
DNA and the DNA to be inserted with either the
