Gene Cloning

Gene Cloning in the Functional Analysis of Proteins 305
and gene knockouts. In this section, we will look at how cloning methods can
be used to produce mutations at a much finer level than this. In fact, as you
will see, it is possible to change any base in any DNA sequence to any other
base, which means that we can also change any amino acid in any protein to
any other amino acid. This method is known as site-directed mutagenesis,
since the mutation is engineered at a precisely predefined site in the DNA.
The methodology of site-directed mutagenesis
The principle behind site-directed mutagenesis is simple, although (as we
shall see) technical fixes are required for it to work at high efficiency. To
explain this principle, we will start by describing the original method;
refinements will be discussed subsequently. Figure 10.13 shows a gene,
1
C
A
Anneal mutagenic primer
to single-stranded plasmid
containing the target gene
2
C
A
Extend primer with
DNA polymerase, seal
gap with DNA ligase
3
Transform
into E. coli
C
G
T
A
Two populations of plasmids result: wildtype,
and with A:T to G:C substitution
Figure 10.13 The basics of site-directed mutagenesis. An oligonucleotide,
complementary to the target gene except for the altered base at the desired
position of the mutation, is annealed to a single-stranded plasmid containing the
gene that we wish to mutate 1). DNA synthesis (catalyzed by DNA polymerase)
of the second strand is primed by the oligonucleotide, and the new strand is
sealed to 3′ end of the primer by DNA ligase 2). When transformed into E. coli,
two plasmids will result as the plasmid replicates: one wild-type and one mutated
at the desired position 3). The plasmids can be distinguished by sequencing.