Processes of Recombinant DNA Technology
Recombinant DNA technology involves several steps in specific sequence. These steps are as follows:
- Iisolation of DNA,
- Fragmentation of DNA by restriction endonucleases,
- Isolation of a desired DNA fragment,
- Ligation of the DNA fragment into a vector,
- Transferring the recombinant DNA into the host,
- Culturing the host cells in a medium at large scale
- Extraction of the desired product.
Isolation of the Genetic Material (DNA)
- The cell is first treated with suitable enzymes to break the membrane; like cell membrane or cell wall.
- Now, RNA is removed by treatment with ribonuclease. Protein is removed by treatment with protease. Other molecules are removed by suitable treatments. After that, addition of chilled ethanol facilitates the precipitation of purified DNA. This can be seen as collection of fine threads in the suspension.
Cutting of DNA at Specific Locations
Purified DNA molecules are incubated with restriction enzyme. Optimal conditions are maintained for a particular enzyme. The progress of a restriction enzyme digestion is checked with the help of Agarose gel electrophoresis. As DNA is negatively charged, it moves towards the positive electrode (anode). This process is repeated with the vector DNA as well.
The cut portion from the source DNA and the cut vector are mixed and ligase is added. This results in the preparation of recombinant DNA.
Amplification of Gene of Interest using PCR
PCR stands for Polymerase Chain Reaction. Multiple copies of the gene of interest are synthesized in vitro during this reaction. It is achieved by using two sets of primers and the enzyme DNA polymerase. (Small chemically synthesized oligonucleotides which are complementary to the regions of DNA are called primers).
Insertion of Recombinant DNA into the Host Cell/Organism
A ligated DNA can be introduced into recipient cells by many methods.
Obtaining the Foreign Gene Product
The ultimate aim of recombinant DNA technology is to produce a desirable protein in large quantities. This can be done on a small scale in laboratory; by using continuous culture system. In this system, the used medium is drained out from one side, and fresh medium is added from the other to maintain the cells in their physiologically most active log/exponential phase.
For producing large volumes of culture, bioreactors are used. Stirring type bioreactors are the most commonly used bioreactors. It is generally cylindrical with a curved base. The stirrer facilitates even mixing and oxygen availability through the reactor. A good quality bioreactor has an agitator system, a temperature control system, pH control system and sampling ports.
Downstream Processing
Downstream processing involves separation and purification of the finished product. If the finished product is a drug, it has to undergo thorough clinical trials. Strict quality control measures are also required.