Once the genomic library has been generated it is necessary to screen for the gene of interest within thousands of recombinant clones. The choice of screening method will very much depend on the availability of reagents and the information on the target gene to be isolated.
Once the
genomic library has been generated it is necessary to screen for the gene of interest
within thousands of recombinant clones. The choice
of screening method will very much depend
on the availability of reagents and the information on the target
gene to be isolated.
This technique is used when
some of the DNA sequence
for the gene screened for is known,
or when a fragment of this gene is available from a previous
cloning. Alternatively, a DNA fragment from
a closely related
gene can be used as a probe
for the isolation of the gene
of interest. The hybridization technique
requires plating the library on a set of agar plates
to generate a replica, on nitrocellulose or nylon membranes, of the
plaques or colonies, each containing a different recombinant DNA fragment. This process
transfers a portion
of each plaque or colony to the membranes and is done in such
a way that the pattern
of plaques/colonies on the original
plate is maintained on the filters.
The membranes are then
hybridized with a radiolabelled DNA probe containing
part of the sequence to be isolated
from the library.
The probe will only bind/hybridize to the recombinant clones containing that sequence. After this process,
the membranes are exposed to X-ray film
(autoradiography). The
presence of dark spots on the films
represents the location
of colonies containing the target gene.
By orienting the film with the original agar plate, the colony/plaque
carrying the complementary sequence can be identified and the desired clone
isolated. An alternative to the use of radio-isotopes for the probes
resides in the use of nucleotides labelled with a molecule such as digoxigenin (DIG). In that
case detection of the hybridized probes is performed
with anti-DIG antibodies conjugated to an enzyme such as alkaline phosphatase, which reacts with a substrate to produce chemiluminescence.
This technique is used when we need to isolate a gene coding for a protein for which there are antibodies available. The success of this technique relies on the expression of the gene of interest, as it requires the synthesis of the target protein from the target recombinant gene. The screening steps are similar to those used for the hybridization screening, with the difference that the membranes containing portions of plaques or colonies have to be incubated with the antibodies that will recognize the target protein. This antibody, called the primary antibody, will bind tightly to those colonies/plaques containing the recombinant gene of interest, provided that the protein encoded by this gene has been synthesized. The position of the bound antibody is revealed by incubating the membranes with a labelled antibody (secondary antibody) that recognizes the primary antibody. There are different types of labels for antibodies, all of which can easily be detected.
This type of screening is limited to proteins that have a specific activity that can easily
be identified within
a large population of recombinant clones. Needless to say, to detect a protein activity the gene coding
for this protein must be expressed and an active protein must be produced. Understanding of this technique can be helped
by illustrating this screening with an example. Suppose we want to isolate
a gene coding for a bacterial haemolytic toxin from a genomic
library. As we know that
this toxin lyses red blood
cells, we could
plate the library
on plates containing agar mixed with blood. Those colonies/ plaques expressing the haemolytic toxin
could easily be identified by the presence
of a haemolytic halo around them resulting from the action
of the toxin on the red
blood cells.
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