that makes M13 useful in chain termination sequencing is the placement of universal
priming sites at20 or40 bases from the start of the MCS. This allows any gene to
be sequenced by using one universal primer since annealing of the primer prior to
sequencing occurs outside the MCS and so is M13-specific rather than gene-specific.
This obviates the need to synthesise new oligonucleotide primers for each new foreign
DNA insert. A further, reverse priming site is also located at the opposite end of the
polylinker allowing sequencing in the opposite orientation to be undertaken.6.6.2 In vitromutagenesis
One of the most powerful developments in molecular biology has been the ability to
artificially create defined mutations in a gene and analyse the resulting protein following
in vitroexpression. Numerous methods are now available for producingsite-directed
mutationsmany of which now involve the PCR. Commonly termedprotein engineering,
this process involves a logical sequence of analytical and computational techniques
centred around a design cycle. This includes the biochemical preparation and analysis
of proteins, the subsequent identification of the gene encoding the protein and its
modification. The production of the modified protein and its further biochemical analysis
completes the concept of rational redesign to improve a protein’s structure and function
(Fig. 6.31).
The use of design cycles and rational design systems are exemplified by the study
and manipulation of subtilisin. This is a serine protease of broad specificity and of
considerable industrial importance being used in soap powder and in the food and
leather industries. Protein engineering has been used to alter the specificity, pH profile
and stability to oxidative, thermal and alkaline inactivation. Analysis of homologous
thermophiles and their resistance to oxidation has also been improved. Engineered
subtilisins of improved bleach resistance and wash performance are now used in many
brands of washing powders. Furthermore mutagenesis has played an important role in
the re-engineering of important therapeutic proteins such as the Herceptin antibody
which has been used to successfully treat certain types of breast cancer.6.6.3 Oligonucleotide-directed mutagenesis
The traditional method of site-directed mutagenesis demands that the gene be already
cloned or subcloned into a single-stranded vector such as M13. Complete sequencing
of the gene is essential to identify a potential region for mutation. Once the precise
base change has been identified an oligonucleotide is designed that is complementary
to part of the gene but has one base difference. This difference is designed to alter a
particular codon, which, following translation, gives rise to a different amino acid and
hence may alter the properties of the protein.
The oligonucleotide and the single-stranded DNA are annealed and DNA polymer-
ase is added together with the dNTPs. The primer for the reaction is the 3^0 end of the
oligonucleotide. The DNA polymerase produces a new complementary DNA strand to
the existing one but which incorporates the oligonucleotide with the base mutation.
The subsequent cloning of the recombinant produces multiple copies, half of which231 6.6 Applications of gene cloning