9780521516358book.pdf

from each reaction to hybridise. Following this, one of the two hybrids bearing a free

30 hydroxyl group is extended to produce a new duplex fragment. The other hybrid with

a5^0 hydroxyl group cannot act as substrate in the reaction. Thus, the overlapped and

extended product will now contain the directed mutation (Fig. 6.33). Deletions and

insertions may also be created with this method although the requirements of four

primers and three PCR reactions limits the general applicability of the technique.

A modification of the overlap extension PCR may also be used to construct directed

mutations; this is termedmegaprimer PCR. This method utilises three oligonucleotide

primers to perform two rounds of PCR. A complete PCR product, the megaprimer is made

single-stranded and this is used as a large primer in a further PCR reaction with an

additional primer.

The above are all methods for creating rational defined mutations as part of a

design cycle system. However it is also possible to introduce random mutations into a

gene and select for enhanced or new activities of the protein or enzyme it encodes.

This accelerated form of artificial molecular evolution may be undertaken using

Oligonucleotide primer

with predefined mutation

Single-stranded

DNA template (M13)

DNA

polymerase

Transform

E. coli

Complementary strand

synthesis with primer incorporated

(with mutation)

Wild-type original strand Strand with new mutation

Anneal primer to template

Fig. 6.32Oligonucleotide-directed mutagenesis. This technique requires a knowledge of nucleotide sequence,

since an oligonucleotide may then be synthesised with the base mutation. Annealing of the oligonucleotide to

complementary (except for the mutation) single-stranded DNA provides a primer for DNA polymerase to

produce a new strand and thus incorporates the primer with the mutation.

233 6.6 Applications of gene cloning