1-2.2.9Screening for expression:

• Gene expression involves the synthesis of mRNA through transcription followed by synthesis of protein through translation. If the purpose of cloning is to express a foreign gene, it is necessary to check the expression both at the mRNA and protein level in terms of quality and quantity.
  

• Screening for foreign gene mRNA transcript can be done by:

► Northern blotting: It involves the electrophoresis for the separation of RNA on the basis of size, and then transfer of RNA from the electrophoresis gel to the blotting membrane. It is then detected by a hybridization probe complementary to target sequence.


► Reverse transcriptase PCR: The RNA strand is reverse transcribed into its DNA complement (cDNA) using the enzyme reverse transcriptase and the resulting cDNA are then amplified using routine PCR method.

• Screening for foreign protein is done by:

►   Western blotting: It involves the process of electrophoretic separation of cellular proteins followed by transfer to a blotting membrane, incubation with a complimentary primary antibody probe and detection with a labelled secondary antibody.


► Activity/functional testing assay or staining: The proteins which have specific functionality or activity can be tested in-vitro to confirm the presence of the same. For example, protease activity testing or staining.

Quantitative evaluation of the expression levels of the protein is necessary to achieve desired amount of protein.