Gene Cloning

Gene Cloning in the Functional Analysis of Proteins 307
A
T
G
C
A
G
Anneal primers
C
T
Extend in PCR
A
G
C
T
Repeated rounds of PCR
C
G
A A C
T G T
Does not cut
with DpnI
Cut with DpnI
Transform into E. coli:
all colonies should carry
desired mutation
Figure 10.14 One method for high efficiency site-directed mutagenesis.
Two primers are produced which are complementary to each other and to the
target gene, but containing the desired mutation. In the first round of a PCR
reaction using these two primers, plasmids with a single mismatch and a single
nicked strand (as no DNA ligase is present) will be produced. Subsequent PCR
reactions will lead predominantly to a double-stranded plasmid with nicks on
both strands and the new base present on both strands. Digestion with DpnI
before transformation removes plasmid molecules containing non-mutated bases.
transformed into E. coli, most of the mutated strands will be converted
back to wild type. Over the years that site-directed mutagenesis has been
in use as a technique, there have been many methods devised to get round
this problem. Below, and in Figure 10.14, we describe one of them which
is currently widely used.
In this method, two primers are used rather than one. The two primers
are complementary to one another and also to the DNA to be mutated,
except that they contain the desired mutation. These primers are mixed