Genetic Manipulation 219flanked by DNA whose sequence is known or at least a close approximation can
be guessed. This knowledge allows a short sequence of DNA to be synthesised
of only a few nucleotides long, to bind specifically to the end of the sequence and
act as a primer for the DNA polymerase to make one copy of the whole piece
of DNA. A second probe is used for the other end to allow the second strand to
be synthesised. The process is repeated by a constant cycling of denaturation of
double-stranded DNA at elevated temperature to approximately 95◦C, followed
by cooling to approximately 60◦C to allow annealing of the probe and comple-
mentary strand synthesis. This technique requires the use of DNA polymerases
able to withstand such treatment and two bacteria from which polymerases have
been isolated for this purpose areThermococcus litoralisandThermus aquaticus.
This latter extremophile has been discussed in Chapter 3.
Cloning vectors
A cloning vector is frequently a plasmid or a bacteriophage (bacterial virus)
which must be fairly small and fully sequenced, able to replicate itself when
reintroduced into a host cell, thus producing large amounts of the recombinant
DNA for further manipulation. Also it must carry on it ‘selector marker’ genes.
These are different from the reporter genes described below which are indicators
of genomic integrity and activity. A common design of a cloning vector is one
which carries two genes coding for antibiotic resistance. The ‘foreign’ gene is
inserted within one of the genes so that it is no longer functional therefore it is
possible to discriminate by standard microbiology techniques which bacteria are
carrying plasmids containing recombinant DNA and which are not. Selector genes
may operate on at least two levels, the first at the level of the bacterium, usually
Eschericia coli, in which the manipulations are being performed described above
and the second being at the level of the final product, for example, a higher plant.
In this case such a selector gene can be resistance to antibiotics like kanamycin
or hygromycin.
Standard cloning vectors normally carry only selector marker genes required
for plasmid construction. To make the manipulations easier, these genes nor-
mally contain a multicloning site (MCS) which is a cluster of sites for restriction
enzymes constructed in such a way to preserve the function of the gene. Disrup-
tion by cloning into any one of these sites will lose the function of that gene and
hence, for example, if it codes for antibiotic resistance, will no longer protect
the bacterium from that antibiotic. An example is shown in Figure 9.2. This is
pGEM(Promega 1996) which has a MCS in the ß-galgene. This codes for
ß-galactosidasefrom theE. coli lacoperon, which has the capacity to hydrolyse
x-gal, a colourless liquid, to produce free galactose and ‘x’ which results in a blue
pigment to the colony. Thus the screening for successful insertion into the MCS
is a simple scoring of blue (negative) or white (possibly positive) colonies. The
success of the experiment can be determined quickly as this cloning vector also
has sequences at either side of the MCS which allows for rapid DNA sequencing.