8 - Selection, screening and analysis of recombinants

Summary

In this final chapter of Part II, the various techniques that can be used to identify cloned genes will be described. As with previous chapters, the basis of techniques that are perhaps not so widely used today will be included, to illustrate the principles of gene identification and characterisation. This will lead into the final section of the book, where various applications of the technology will be covered, and where we get a look at some of the more advanced developments in gene manipulation.

Success in any cloning experiment depends on being able to identify the desired gene sequence among the many different recombinants that may be produced. Given that a large genomic library may contain a million or more cloned sequences, whic h are not readily distinguishable from each other by simple analytical methods, it is clear that identification of the target gene is potentially the most difficult part of the cloning process. Fortunately there are several selection/identification methods that can be used to overcome most of the problems that arise.

There are two terms that require definition before we proceed, these being selection and screening. Selection is where some sort of pressure (e.g. the presence of an antibiotic) is applied during the growth of host cells containing recombinant DNA. The cells with the desired characteristics are therefore selected by their ability to survive. This approach ranges in sophistication, from simple selection for the presence of a vector, up to direct selection of cloned genes by complementation of defined mutations.