Screening of Genomic Libraries

SCREENING OF GENOMIC LIBRARIES

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.

A)   Hybridization Screening

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.

B)  Immunological Screening

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.

C) Protein Activity Screening

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.