superior to cloned double-stranded probes indicated in Section 6.4.1. They are used
extensively inin situhybridisation and for identifying and analysing mRNA and are
described in more detail in Section 6.8.6.5 Screening gene libraries
6.5.1 Colony and plaque hybridisation
Once a cDNA or genomic library has been prepared the next task requires the identifi-
cation of the specific fragment of interest. In many cases this may be more problematic
than the library construction itself since many hundreds of thousands of clones may be
in the library. One clone containing the desired fragment needs to be isolated from the
library and therefore a number of techniques mainly based on hybridisation have been
developed.
Colony hybridisationisonemethodusedtoidentifyaparticularDNAfragmentfroma
plasmid gene library (Fig. 6.27). A large number of clones are grown up to form colonies
on one or more plates, and these are then replica plated onto nylon membranes placed on
solid agar medium. Nutrients diffuse through themembranes and allow colonies to grow
on them. The colonies are then lysed, and liberated DNA is denatured and bound to the
membranes, so that the pattern of colonies is replaced by an identical pattern of bound
DNA. The membranes are then incubated with a prehybridisation mix containing non-
labelled non-specific DNA such as salmon sperm DNA to block non-specific sites.
Following this denatured, labelled gene probe is added. Under hybridising conditions
the probe will bind only to cloned fragments containing at least part of its corresponding
gene (Section 5.9.3). The membranes are then washed to remove any unbound probe and
the binding detected by autoradiography of the membranes. If non-radioactive labels
have been used then alternative methods of detection must be employed (Section 5.9.4).
By comparison of the patterns on the autoradiograph with the original plates of colonies,
those that contain the desired gene (or part of it) can be identified and isolated for further
analysis.Asimilarprocedureisusedtoidentifydesiredgenesclonedintobacteriophage
vectors. In this case the process is termedplaque hybridisation. It is the DNA contained in
the bacteriophage particles found in each plaque that is immobilised on to the nylon
membrane.Thisisthenprobedwithanappropriatelylabelledcomplementarygeneprobe
and the detection undertaken as for colony hybridisation.6.5.2 PCR screening of gene libraries
In many cases it is now possible to use the PCR to screen cDNA or genomic libraries
constructed in plasmids or bacteriophage vectors. This is usually undertaken with
primers which anneal to the vector rather than the foreign DNA insert. The size of an
amplified product may be used to characterise the cloned DNA and subsequent
restriction mapping is then carried out (Fig. 6.28). The main advantage of the PCR
over traditional hybridisation based screening is the rapidity of the technique, as PCR225 6.5 Screening gene libraries