as well as naturally existing ones. This strategy is not restricted to antibodies and vast
libraries of peptides may be used in this combinatorial chemistry approach to identify
novel compounds of use in biotechnology and medicine.
Phage-based cloning methods also offer the advantage of allowing mutagenesis to
be performed with relative ease. This may allow the production of antibodies with
affinities approaching that derived from the human or mouse immune system. This
may be brought about by using an error prone DNA polymerase in the initial steps of
constructing aphage display library. It is possible that these types of libraries may
provide a route to high affinity recombinant antibody fragments that are difficult to
produce by more conventional hybridoma fusion techniques. Surface display libraries
have also been prepared for the selection of ligands, hormones and other polypeptides
in addition to allowing studies on protein–protein or protein–DNA interactions or
determining the precise binding domains in these receptor–ligand interactions.6.7.3 Alternative display systems
A number of display systems have been developed based on the original phage display
technique. One interesting method isribosome displaywhere a sequence or even a library
of sequences are transcribed and translatedin vitro. However in the DNA library the
sequences are fused to spacer sequences lacking a stop codon. During translation at
the ribosome the protein protrudes from the ribosome and is locked in with the mRNA.
The complex can be stabilised by adding salt. In this way it is possible to select the
appropriate protein through binding to its ligand. Thus a high-affinity protein-ligand
can be isolated which has the mRNA that originally encoded it. The mRNA may then be
reverse transcribed into cDNA and amplified by PCR to allow further methods such as
mutagenesis to be undertaken. A related technique,mRNA display, is similar except the
association between the protein and mRNA is through a more stable covalent puromycin
link rather than the salt-induced link as in ribosome display. Further display systems,
based on yeast or bacteria, have also been developed and provide powerfulin vitro
selection methods.6.8 Analysing genes and gene expression
6.8.1 Identifying and analysing mRNA
The levels andexpression patterns ofmRNA dictate many cellularprocessesandtherefore
there is much interest in the ability to analyse and determine levels of a particular mRNA.
Technologies such as real-time orquantitative PCRand microchip expression arrays are
currently being employed and refined for high throughput analysis. A number of other
informative techniques have been developed that allow the fine structure of a particular
mRNA to be analysed and the relative amounts of an RNA quantitated by non-PCR-based
methods. This is important not only for gene regulation studies but may also be used as a
marker for certain clinical disorders. Traditionally the Northern blot has been used for240 Recombinant DNA and genetic analysis