Recombinant DNA technology involves several steps in specific sequence such as isolation 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 and extraction of the desired product.
❯ Isolation of DNA
DNA is isolated by first degrading all the membranes enclosing it with specific enzymes
For bacterial cell – Lysozyme
For plant cells – Cellulase
For fungal cells – Chitinase
RNA is removed by treatment with RNA digesting enzymes – Ribonuclease.
Proteins are removed by treatment with protein digesting enzymes – Protease.
Other biomolecules are removed by appropriate treatments.
Finally, the DNA is precipitated out by the addition of chilled ethanol.
This is seen as collection of fine threads in the suspension.
This DNA can be removed by spooling.
❯ Cutting of DNA at Specific Locations
DNA can be cut at specific location by digesting the purified DNA with specific Restriction enzyme.
Agarose gel electrophoresis can be used to check the progress of restriction digestion.
After the source DNA and the vector DNA have been cut with a specific RE, the gene of interest is cut out and is ligated with the cut vector DNA (plasmid).
The ligation of the gene of interest and the vector DNA is mediated by an enzyme named – DNA ligase.
❯ Amplification of Gene of Interest using PCR
The Polymerase Chain Reaction (PCR) results in the selective amplification of a chosen region of a DNA molecule.
✼ Requirements for PCR
Template DNA
Deoxynucleotides/deoxyribonucleotides
Thermostable enzyme: Taq Polymerase
Source: Bacterium – Thermus aquaticus
Property: The enzyme remains active during the high temperature.
Sets of Primers:
Primers are small chemically synthesized oligonucleotides that are complementary to the regions of DNA.
They delimit the region of DNA to be amplified.
✼ Step involved in PCR
PCR is carried out in a single test tube simply by mixing DNA with a set of reagents and placing the tube in a thermal cycler.
Denaturation
The mixture is heated to 94°C, at which temperature the hydrogen bonds that hold together the two strands of the double-stranded DNA molecule are broken, causing the molecule to denature.
It forms single stranded DNA (ssDNA).
Annealing
The mixture is cooled down to 50–60°C.
The primers anneal (joins) with the ssDNA molecules at specific positions.
Polymerase Chain Reaction
Extension
The temperature is raised to 74°C.
The Taq DNA polymerase works best at this temperature.
It attaches to one end of each primer and synthesizes new strands of DNA, complementary to the template DNA molecules.
This results in four stands of DNA instead of the two that there were to start with.
❯ Insertion of Recombinant DNA into the Host Cell/Organism
Recipient cells after making them ‘competent’ to receive, take up DNA present in its surrounding.
So, if a recombinant DNA bearing gene for resistance to an antibiotic (e.g., ampicillin) is transferred into E. coli. cells, the host cells become transformed into ampicillin-resistant cells.
If we spread the transformed cells on agar plates containing ampicillin, only transformants will grow, untransformed recipient cells will die.
Since, due to ampicillin resistance gene, one is able to select a transformed cell in the presence of ampicillin. The ampicillin resistance gene in this case is called a selectable marker.
❯ Obtaining the Foreign Gene Product
In most of the recombinant technologies, the ultimate aim is to produce a desirable protein.
The foreign gene gets expressed and produces the maximum protein in appropriate conditions.
Recombinant protein: If any protein encoding gene is expressed in a heterologous host.
✼ Culturing of Host cell
The host cells with the rDNA is grown on an appropriate condition (proper nutrients, temperature, pH, etc.)
Small scale culture: Cells are grown in the laboratory in cultures. The protein are then extracted and purifies by using different separation techniques.
Large scale culture: Cells are grown in continuous culture system wherein the used medium is drained out from one side while fresh medium is added from the other to maintain the cells in their physiologically most active log/exponential phase.
✼ Bioreactors
These are large vessels of large volumes (100-1000 litres), in which raw materials are biologically converted into specific products, individual enzymes, etc., using microbial, plant, animal or human cells.
It provides the optimal conditions for achieving the desired product by providing optimum growth conditions (temperature, pH, substrate, salts, vitamins, oxygen).
✼ Components of a bioreactor:
an agitator system
an oxygen delivery system
a foam control system
a temperature control system
pH control system
sampling ports so that small volumes of the culture can be withdrawn periodically
Stirred Tank Bioreactor
A stirred-tank reactor is usually cylindrical or with a curved base to facilitate the mixing of the reactor contents.
The stirrer facilitates even mixing and oxygen availability throughout the bioreactor.
Sparged Stirred Tank Bioreactor
It is a stirred-tank reactor type bioreactor where the air is bubbled.
Bioreactors
❯ Downstream processing
It involves all the stages after the expression of the gene product in the culture system by the host cells (biosynthetic stage).
The processes include separation and purification, which are collectively referred to as downstream processing.
Suitable preservatives if required are added.
If the product is a drug, then it undergoes clinical trials.
The downstream processing and quality control testing vary from product to product.
