Explain the steps in process of rDNA technology with suitable diagrams.
The steps involved in gene cloning are as follows:
i. Isolation of DNA (gene) from the donor organism:
- The desired gene to be cloned is obtained from the source organism (donor).
- Initially, the cells of the donor organism are sheared with the blender and treated with a suitable detergent.
- Genetic material from the donor is isolated and purified using several techniques.
- Isolated DNA can be spooled onto a glass rod.
ii. Cutting of the desired gene:
- Isolated purified DNA is then cleaved by using restriction enzymes i.e. restriction endonucleases.
- These enzymes cleave DNA at restriction sites and break the DNA into fragments.
- There are several types of restriction endonucleases.
- Cleaved DNA fragments have cohesive, sticky, staggered ends or blunt ends.
- From cleaved DNA fragments, a fragment containing the desired gene is isolated and selected for cloning. This is now called foreign DNA or passenger DNA.
- The desired gene can also be obtained directly from the genomic library or cDNA library.
iii. Insertion of a desired foreign gene into a cloning vector (vehicle DNA):
- The foreign DNA or passenger DNA is now inserted into a cloning vector or vehicle DNA.
- The most commonly used cloning vectors are plasmids of bacteria and bacteriophage viruses like lambda phage and M13.
- The most commonly used plasmid is pBR322.
- Plasmids are isolated from the vector organisms i.e. bacterium.
- By using the same restriction enzyme (which is used in the isolation of the desired gene from the donor), plasmid i.e. vector DNA is cleaved.
- Now by using enzyme DNA ligase, foreign DNA is inserted/ integrated into the vector DNA.
- The combination of vector DNA and foreign DNA is now called Recombinant DNA or Chimeric DNA and the technology is referred to as rDNA technology.
iv. Transfer of rDNA into suitable competent host or cloning organism:
- Finally, the recombinant DNA is transferred for expression into a competent host cell which is usually a bacterium.
- The host cell takes up naked rDNA by process of ‘transformation’ and incorporates it into its own chromosomal DNA which finally expresses the trait controlled by passenger DNA.
- The transfer of rDNA into a bacterial cell is assisted by divalent Ca++.
- The cloning organisms used in plant biotechnology are E. coli and Agrobacterium tumefaciens.
- The host/ competent cell which has taken up rDNA is now called a transformed cell.
- Foreign DNA can also be transferred directly into the naked cell or protoplast of the competent host cell, without using a vector.
- This is done by using techniques like electroporation, microinjection, lipofection, shotgun, ultra-sonification, biolistic method, etc. But in plant biotechnology, the transformation is through Ti plasmids of A. tumefaciens.
v. Selection of the transformed host cell:
- The transformation process generates a mixed population of transformed (recombinant) and non-transformed (non-recombinant) host cells.
- For the isolation of recombinant cells from non-recombinant cells, the marker gene of the plasmid vector is employed.
- For example, the pBR322 plasmid vector contains different marker genes (Ampicillin resistant gene and Tetracycline resistant gene).
- When the PstI restriction enzyme is used, it knocks out Ampicillin resistant gene from the plasmid, so that the recombinant cell becomes sensitive to Ampicillin.
vi. Multiplication of transformed host cell:
- Once transformed, host cells are separated by the screening process.
- In this step, the transformed host cells are introduced into fresh culture media.
- At this stage, the host cells multiply along with the replication of the recombinant DNA carried by them.
vii. Expression of the gene to obtain the desired product:
- The next step involves the production of desired products like alcohol, enzymes, antibiotics, etc.
- Finally, the desired product is separated and purified through downstream processing using a suitable bioreactor.
Outline of the process of recombinant DNA technology