Biomedical Engineering Reference
In-Depth Information
For a detailed review of phage and phagemid dis-
play, the reader should consult Sidhu (2000) and
Sidhu
et al.
(2000).
fragments carrying the desired mutation and the
homologous region on the chromosome.
Unlike many organisms,
E. coli
is not readily
transformed by linear DNA fragments. The main
reason for this is the rapid degradation of the DNA by
the intracellular RecBCD exonuclease. Mutant
strains lacking the exonuclease do not degrade lin-
ear DNA very rapidly, but such strains grow poorly,
are defective for recombination and do not support
efficient replication of plasmid vectors. The solution
devised by Yu
et al.
(2000) was to use an
E. coli
strain
containing a
Directed mutation
in vivo
In vitro
mutagenesis methods are very useful when
working with small plasmids carrying cloned genes.
However, if it is desired to modify a single residue on
a very large vector, such as a yeast artificial chro-
mosome (YAC), P1-derived artificial chromosome
(PAC) or bacterial artificial chromosome (BAC),
in vitro
methods are not appropriate. With such
large plasmids, directed mutagenesis has to be done
in vivo
and a number of different methods have
been described (Muyrers
et al.
2000, Lalioti &
Heath 2001). The simplest is that of Yu
et al.
(2000),
which involves recombination between linear DNA
prophage harbouring the genes
exo
,
bet
and
gam
under the control of a temperature-
sensitive
λ
cI repressor. The Gam gene product stops
the RecBCD nuclease from attacking the linear
DNA and the Exo and Bet gene products generate
recombination activity to enable DNA exchange
to occur.
λ
