Recombinant DNA technology is the technique of combining distinct genetic materials (DNA) and introducing them into host organisms from two different species or sources to create new genetic combinations.
Medicine, science, industry, and agriculture all benefit from these new combinations.
The task of inserting a gene into the host genome is not simple.
The appropriate gene must first be chosen for injection into the host, followed by a suitable vector for forming recombinant DNA by integrating the gene with the vector.
Herbert Boyer and Stanley Cohen used E.Coli restriction enzymes to introduce foreign DNA from plasmids into living organisms, resulting in recombinant DNA.
Tools used in the recombinant DNA technology process
- Restriction Enzymes: They identify the site in the vector genome where the desired gene is introduced.
Endonucleases are enzymes that cleave DNA strands.
Exonucleases are enzymes that remove nucleotides from the end of a DNA strand.
- Enzyme Ligase: This enzyme joins two fragments together. The ligase enzyme is used to join the sticky ends of the desired gene with the vector.
- Vector: A plastid plasmid is typically used to carry and integrate the desired gene.
- Host: A capable host cell into which the recombinant DNA is implanted.
Steps in the DNA Technology Process
1. Genetic Material Isolation
Isolation of required DNA in its purest form, that is, without the presence of other macromolecules.
Within the cell membrane of a typical cell, DNA coexists with other macromolecules such as proteins, RNA, and polysaccharides.
It must be purified and separated from other macromolecules using enzymes such as cellulose, Lysozyme, Chitinase, proteases, and ribonuclease.
The addition of ethanol causes DNA to precipitate out as a fine thread, and refined DNA is pulled out, a process known as spooling.
2. Enzyme Digestion with Restriction
Restriction enzyme digestion is a procedure in which restriction enzymes, which serve as ‘Molecular Scissors,’ cut DNA at a specific spot.
The purified DNA is subsequently treated with the restriction enzyme of choice under optimal conditions for that enzyme.
Agarose gel electrophoresis is a technique that uses an Agarose gel to separate the DNA.
With the assistance of current, Because DNA is negatively charged, it flows to the positive electrode and is segregated based on its size.
This makes it possible to isolate and clip out digested DNA pieces. Vector DNA can also be utilised with the same technique.
3. PCR-based amplification
The polymerase chain reaction (PCR) is a method for making numerous copies of a DNA sequence in vitro using the enzyme DNA polymerase.
By amplifying a single copy of DNA with the help of PCR, millions of copies of DNA can be created.
Thermal cyclers require the following components to execute PCR reactions.
The template is the DNA that will be amplified.
Primers are short oligonucleotides that are chemically produced and complementary to a certain DNA sequence.
Enzyme: DNA polymerase is a protein that breaks down DNA. Nucleotides are required for the enzyme to extend primers. The cut DNA fragments can be amplified with PCR and then ligated with cut vectors.
DNA Recombinant Host Insertion
In this step, there is a metamorphosis. It is a procedure in which recombinant DNA is introduced into a recipient cell, usually a bacterial cell that has been rendered competent to accept additional DNA.
Ca+2 ion therapy, thermal shock, electroporation, and other procedures are utilised to make a cell competent. 6.
Obtaining and Culturing Foreign Gene Products A bit of alien DNA is introduced into a cloning vector, and the alien DNA is duplicated in a bacterial cell.
The ultimate goal is to produce desired protein expression. The protein encoded by the encoded gene and expressed in the heterologous host is referred to as recombinant protein.
To create a big amount of recombinant protein that benefits humans, a high volume or cell culture is required.
Bioreactors are the vessels used to do this. Bioreactors are enormous containers that can process anywhere from 100 to 1000 litres of cell culture.
A bioreactor provides optimal conditions, such as pH and temperature, for biologically converting raw materials into specialised enzymes and proteins.
Processing in the Aftermarket
This procedure includes marketing protein as a finished product following quality control, purification, and clinical testing.
Conclusion
Recombinant DNA technology is the technique of combining distinct genetic materials (DNA) and introducing them into host organisms from two different species or sources to create new genetic combinations.
Medicine, science, industry, and agriculture all benefit from these new combinations.
The task of inserting a gene into the host genome is not simple.