Genetic Manipulation - Environmental Biotechnology: Theory and Application - page 217

Polymerase chain reaction
The PCR is a powerful technique which amplifies a piece of DNA of which
only a very few copies are available. The piece must be flanked by DNA whose
sequence is known or at least a close approximation can be guessed. This 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 ele-
vated temperature to approximately 95 ◦ C, followed by cooling to approximately
60 ◦ C to allow annealing of the probe and complementary strand synthesis. This
technique requires the use of DNA polymerases able to withstand such treat-
ment. Two bacteria from which polymerases have been isolated for this purpose
are Thermococcus litoralis and Thermus 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 indica-
tors 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 there-
fore 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 Escherichia coli , in which the manipulations are being per-
formed 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 one to confer
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 normally
contain a multi cloning site (MCS) which is a cluster of sites for restriction
enzymes constructed in such a way to preserve the function of the gene. Dis-
ruption 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 β -gal gene. This codes for
β -galactosidase from the E. coli lac operon, which has the capacity to hydrolyse