Biomedical Engineering Reference
In-Depth Information
Single-stranded
M13 recombinant
Chemically synthesized
oligonucleotide
*
*
Anneal
TCAGGCT
(1) DNA polymerase + 4dNTPs
(2) T4 DNA ligase + ATP
*
*
Transform
E. coli
Wild-type
Mutant
*
*
Fig. 7.9
Oligonucleotide-directed
mutagenesis. Asterisks indicate
mismatched bases. Originally the
Klenow fragment of DNA polymerase
was used, but now this has been largely
replaced with T7 polymerase.
Screen plaques with
32
P-labelled
oligonucleotide as hybridization
probe
Isolate mutant
The synthetic oligonucleotide primes DNA syn-
thesis and is itself incorporated into the resulting
heteroduplex molecule. After transformation of the
host
E. coli
, this heteroduplex gives rise to homodu-
plexes whose sequences are either that of the original
wild-type DNA or that containing the mutated base.
The frequency with which mutated clones arise,
compared with wild-type clones, may be low. In
order to pick out mutants, the clones can be screened
by nucleic acid hybridization (see Chapter 6) with
32
P-labelled oligonucleotide as probe. Under suitable
conditions of stringency, i.e. temperature and cation
concentration, a positive signal will be obtained only
with mutant clones. This allows ready detection of
the desired mutant (Wallace
et al.
1981, Traboni
et al.
1983). In order to check that the procedure has
not introduced other adventitious changes, it is prud-
ent to check the sequence of the mutant directly by
DNA sequencing. This was a particular necessity
with early versions of the technique which made use
of
E. coli
DNA polymerase. The more recent use of
the high-fidelity DNA polymerases from phages T4
and T7 has minimized the problem of extraneous
mutations, as well as shortening the time for copying
