1. Isolation of the genetic material :
The gene of interest is isolated from the nucleus of the donor organism. This involves digestion of cell wall or cell membrane of the donor organism by enzymatic action to obtain pure form of DNA.
2. Cutting of DNA at specific locations
- The pure DNA obtained is cut into several fragments using restriction endonucleases.
- The fragments generated by restriction endonucleases are separated according to their size by gel electrophoresis.
3. Ampiflication of gene of interest using PCR
- Polymerase chain reaction (PCR) is used to generate billions of copies of the desired DNA in vitro. This is called amplification.
- The desired gene is inserted into a suitable vector (Plasmid).
- The DNA ligases join the complementary ends of plasmid DNA with that of desired gene. The hybrid DNA is called recombinant DNA.
4. Insertion of recombinant DNA into host cell or organism
- The recombinant DNA is transformed into a suitable bacterial host (E.coli) by transformation for the expression of the desired gene.
- The cells into which rDNA is inserted are called transformed cells.
- The transformed bacterial cells can be identified using suitable selectable markers.
5. Obtaining the foreign gene product
- The transformed bacterial cells (recombinants) are grown in bioreactor having suitable nutrient medium.
- Bioreactors provide optimum conditions for achieving the desired product by providing optimum growth conditions such as temperature, pH, suitable salts, vitamins, oxygen etc.
- The desired protein is extracted from bacterial cells by separation and purification. These processes constitute downstream processing.
