Preparation of a library for screening by PCR can be done by following ways-

•  The library can be plated as plaques or colonies on agar plates and individually inoculated into the wells of the multi-well plate. However it is a labor intensive process and can lead to bias in favor of larger colonies or plaques.

•  The alternative method involves diluting the library. It involves plating out a small part of the original library (the packaging mix for a phage library, transformation for a plasmid library) and calculating the titer of the library. A larger sample is diluted to give a titer of 100 colonies per mL. Dispensing 100 μL into each well theoretically gives 10 clones in each well. These are then pooled and PCR reactions are carried out with gene-specific primers flanking a unique sequence in the target to identify the wells containing the clone of interest. This method is often used for screening commercially available libraries.

4-5.3. Screening methods based on gene expression

4-5.3.1. Immunological screening

This involves the use of antibodies that specifically recognize antigenic determinants on the polypeptide. It does not rely upon any particular function of the expressed foreign protein, but requires an antibody specific to the protein.

Earlier immunoscreening methods employed radio-labeled primary antibodies to detect antibody binding to the nitrocellulose sheet (Figure 4-5.3.1(a).). It is now superseded by antibody sandwiches resulting in highly amplified signals. The secondary antibody recognizes the constant region of the primary antibody and is, additionally, conjugated to an easily assayable enzyme (e.g. horseradish peroxidase or alkaline phosphatase) which can be assayed using colorimetric change or emission of light using X-ray film (Figure 4-5.3.1(b).).

•  In this technique, the cells are grown as colonies on master plates and transferred to a solid matrix.

•  These colonies are subjected to lysis releasing the proteins which bind to the matrix.

•  These proteins are treated with a primary antibody which specifically binds to the protein (acts as antigen), encoded by the target DNA. The unbound antibodies are removed by washing.

•  A secondary antibody is added which specifically binds to the primary antibody removing the unbound antibodies by washing.

•  The secondary antibody carries an enzyme label (e.g., horse radishperoxidase or alkaline phosphatase) bound to it which converts colorless substrate to colored product. The colonies with positive results (i.e. colored spots) are identified and subcultured from the master plate.