[SagarikaGhosh]

Screening based on properties of protein products of target genes have to be based on expression libraries, i.e., libraries constructed using expression vectors (expression cloning). All expression libraries of higher eukaryotes are, of necessity, cDNA libraries, but those of bacteria and many yeast are usually genomic libraries since few, if any, of their genes have introns, and there is very little spacer DNA between genes.

Generally, a random primer method is used to obtain cDNA; as a result, 5-end sequences are likely to be represented in a higher frequency. In addition, some cDNAs may be abundant and others rare due to the tissue used for library construction. A potential problem of genomic expression libraries of bacteria and yeast is that clones corresponding to a gene may carry termination sequences from the gene located immediately upstream; this can prevent efficient expression of the gen. therefore, DNA fragments used for cloning are smaller in size than the target gene.

In addition, a large enough number of recombinant clones are produced so that there is a reasonable chance for the DNA fragments representing each gene to be cloned in all six possible reading frames; three reading frames in each of the two orientations of the fragments with respect to the promoter (present in the vector).

Given below is a list of possible approaches for expression based screening:

1. Hybrid Arrested Translation: HART is an indirect approach for identification of clones having the desired DNA insert and resulting polypeptide.

2. Hybrid Selection: Recombinant DNAs are fixed to solid support like nitrocellulose filter discs which hybridizes to complementary mRNA which is used for in vitro translation and therefore identification of the DNA.

3. Antibodies Specific to the Protein Product: This is used if the protein lacks a recognizable and measurable function.

4. Colony / Plaque Screening with Antibodies: Antibodies are bound to solid support and brought in contact with agar containing colonies to be screened, proteins produced by the colonies bind to the antibodies and can therefore be identified.

5. Fluorescence Activated Cell Sorter (FACS): This rapid screening method is used for cells whose products are expressed on the cell surface and bind to specific antibodies.

6. Unique Gene Products (Gain of Function): cells can be identified by a function not performed by the proteins of non transformed host cells like enzyme activities or hormone effects.

7. Marker Rescue Approach: Cells are identified by using mutant E.coli strains that produce compounds essential for survival/multiplication of the cells.

8. South-Western Screening: Performed using transcription factors for identification of clones encoding proteins