Biomedical Engineering Reference
In-Depth Information
Multiple point
mutations
Insertion
mutagenesis
Deletion
mutagenesis
Mutant oligonucleotide
with multiple (four)
single base pair
mismatches
Mutant oligonucleotide
carrying a sequence to
be inserted sandwiched
between two regions
with sequences
complementary to sites
on either sides of the
target site in the
template
Mutant oligonucleotide
spanning the region to
be deleted, binding to
two separate sites, one
on either side of the
target
Fig. 7.10
Oligonucleotide-directed
mutagenesis used for multiple point
mutation, insertion mutagenesis and
deletion mutagenesis.
the second strand. Also, these polymerases do not
'strand-displace' the oligomer, a process which would
eliminate the original mutant oligonucleotide.
A variation of the procedure (Fig. 7.10) outlined
above involves oligonucleotides containing inserted
or deleted sequences. As long as stable hybrids are
formed with single-stranded wild-type DNA, prim-
ing of
in vitro
DNA synthesis can occur, ultimately
giving rise to clones corresponding to the inserted or
deleted sequence (Wallace
et al.
1980, Norrander
et al.
1983).
of mutant and non-mutant progeny, but in practice
mutants are counterselected. The major reason for
this low yield of mutant progeny is that the methyl-
directed mismatch repair system of
E. coli
favours
the repair of non-methylated DNA. In the cell, newly
synthesized DNA strands that have not yet been
methylated are preferentially repaired at the position
of the mismatch, thereby eliminating a mutation. In
a similar way, the non-methylated
in vitro
-generated
mutant strand is repaired by the cell so that the
majority of progeny are wild type (Kramer, B.
et al.
1984). The problems associated with the mismatch
repair system can be overcome by using host strains
carrying the
mut
L,
mut
S or
mut
H mutations, which
prevent the methyl-directed repair of mismatches.
A heteroduplex molecule with one mutant and
one non-mutant strand must inevitably give rise to
both mutant and non-mutant progeny upon replica-
tion. It would be desirable to suppress the growth of
non-mutants, and various strategies have been
developed with this in mind (Kramer, B. 1984, Carter
et al.
1985, Kunkel 1985, Sayers & Eckstein 1991).
Another disadvantage of all of the primer exten-
sion methods is that they require a single-stranded
template. In contrast, with PCR-based mutagenesis
(see below) the template can be single-stranded or
double-stranded, circular or linear. In comparison
with single-stranded DNAs, double-stranded DNAs
are much easier to prepare. Also, gene inserts are in
general more stable with double-stranded DNAs.
These facts account for the observation that most
commercial mutagenesis kits use double-stranded
templates.
Deficiencies of the single-primer method
The efficiency with which the single-primer method
yields mutants is dependent upon several factors.
The double-stranded heteroduplex molecules that
are generated will be contaminated both by any
single-stranded non-mutant template DNA that
has remained uncopied and by partially double-
stranded molecules. The presence of these species
considerably reduces the proportion of mutant
progeny. They can be removed by sucrose gradient
centrifugation or by agarose gel electrophoresis, but
this is time-consuming and inconvenient.
Following transformation and
in vivo
DNA syn-
thesis, segregation of the two strands of the het-
eroduplex molecule can occur, yielding a mixed
population of mutant and non-mutant progeny.
Mutant progeny have to be purified away from
parental molecules, and this process is complicated
by the cell's mismatch repair system. In theory, the
mismatch repair system should yield equal numbers
