Screening DNA libraries:

Genomic libraries are screened through hybridization with a DNA probe which is complementary to component of the nucleotide sequence of the desired gene. The probe should be a DNA restriction fragment or perhaps element of a cDNA clone. Alternate approach is possible if some of the protein sequence for the preferred gene is known. While using the genetic code one can then deduce DNA sequence of this category of the gene and synthesize an oligonucleotide with this sequence to act as the DNA probe.

When using a plasmid vector, a simple process for screening would be to take agar plates bearing bacterial colonies which make up the genomic library and overlay every plate with a nitrocellulose membrane which shown in below figure. This is peeled off and is a replica of the plate in which some of the bacterial cells in every colony will have adhered to it and in the similar pattern as the colonies on the plate. This filter is frequently known as a colony lift. That is treated with alkali to lyse the bacterial cells and denature the DNA and then hybridized with a radiolabeled DNA probe. After washing away unreacted probe and autoradiography of the filter shows that

Figure: Screening a gene library by hybridization.

colonies have hybridized with the probe and therefore contain the desired sequences. These are then improved from the agar plate.

When a bacteriophage is used as the cloning vector the gene library is screened as an array of plaques in a bacterial lawn. A hybridization screening technique is used same to which defined for plasmid screening; in this case the replica filter is known as a 'plaque lift'.

For cDNA libraries the screening can as same be carried out by hybridization. Additionally, it is possible to make the cDNA library using a vector that will actually transcribe the inserted cDNA and then translate the resulting mRNA to form protein corresponding to the cloned gene. A library made with like an expression vector is an expression cDNA library. That can be screened using a labeled antibody which recognizes the specific protein and therefore identifies those bacteria that contain the desired gene and are synthesizing the protein. Not just anti- body but any ligand that binds to the goal protein can be used as a probe. For instance, labeled hormone should be used to recognized clones synthesizing hormone receptor proteins.