9780521516358book.pdf

contain a sequence with the mutation and half contain the wild-type sequence. Plaque

hybridisation using the oligonucleotide as the probe is then used at a stringency that

allows only those plaques containing a mutated sequence to be identified (Fig. 6.32).

Further methods have also been developed which simplify the process of detecting the

strands with the mutations.

6.6.4 PCR-based mutagenesis

The PCR has been adapted to allow mutagenesis to be undertaken and this relies on

single bases mismatched between one of the PCR primers and the target DNA to

become incorporated into the amplified product following thermal cycling.

The basicPCR mutagenesissystem involves the use of two primary PCR reactions

to produce two overlapping DNA fragments both bearing the same mutation in the

overlap region. The technique is termedoverlap extension PCR. The two separate PCR

products are made single-stranded and the overlap in sequence allows the products

Protein purification

Protein production

Protein assay

Design Cycle

Protein

Engineering

Site-directed mutations

Protein redesign

& modelling

Copurification of

ligand and protein

Analysis techniques

in solution

Protein

crystallography

Molecular modelling

Comparison with known protein databases

Fig. 6.31Protein design cycle used in the rational redesign of proteins and enzymes.

232 Recombinant DNA and genetic analysis