Recombination DNA technology or rDNA technology is sophisticated molecular biology developed to produce essential biologicals on a wide-scale to match the demands of the huge population.
rDNA technology applications in health care are mind-blowing, for instance, the insulin required as a supplement for diabetic patients was procured previously from cattle and pigs. But the quantity of supply was very small, yet expensive and also not completely suitable to humans due to compatibility (allergic reaction) problems. By rDNA technology, this hindrance was overcome, as insulin which is less expensive and compatible for all the human is produced on such a large-scale to suit the demands of the world market with a continuous supply.
Further enzymes, vaccines, hormones and other biological required can be supplied in large quantities.
Recombinant DNA technology definition:
It is the “process of isolation of the desired gene from an organism of interest and transferring it to an organism of choice to yield the desired product in large quantities.
Explanation: For instance, the Human gene (DNA molecule) coding for insulin is isolated and transferred to E.coli bacteria. Then the cloned bacteria are cultured in suitable media and grown on large-scale. This leads to profuse production of human insulin in large quantities.
Principles of recombinant DNA technology:
Recombination technology has the following steps
- Generation and isolation of the desired genes.
- Insertion of the desired gene into a suitable vector (carrier).
- Transferring the recombinant vector into a host cell.
- Multiplication and separation of cloned cells.
- Expression of the desired gene inside the host cell to get the product.
Generation and isolation of the desired gene: The desired gene is isolated from the whole genome of the organism. For these two enzymes namely “Restriction endonucleases” and “DNA ligases” are used.
Endonucleases are the restriction enzymes that cut the DNA at specific points on the gene so as to get the desired gene. These cut genes are individual single strands and these cut DNA (with sticky ends) are ligated by the help of DNA ligases.
Inserting the desired gene into a vector (carrier):
DNA molecules so isolated are very delicate and get damaged due to travel or transfer. So they require a carrier for their safe transfer.
The isolated DNA is to be inserted into the genome of the cell of a host organism. These vehicles are called “vectors “. They take the desired gene from outside the cell into inside the host cell without any damage. Further, they incorporate it into the host cell genome.
The vectors generally used are
1. Plasmid vectors, 2. Cosmids 3. Bacteriophages 4. Human artificial vectors.
These vectors are large pieces of DNA molecules mostly. The plasmid is a circular, single-stranded and self replicable DNA molecule present inside bacteria. They help in the sexual reproduction of bacteria by transfer of genetic matter from one to another. Here we use them to transfer the desired gene.
While bacteriophage is a virus that attacks bacteria and inserts its gene into the bacterial cell for multiplication. Cosmid is similar to plasmid DNA but can accommodate large DNA pieces.
This insertion of the desired gene into vector DNA is done by using DNA ligase again.
Transferring the recombinant vector into a host cell: The recombinant plasmids (i.e. plasmids with the desired gene) are transferred into host cells.
The host cells suitable for this purpose are
- Prokaryotes: Bacteria like E.coli & Bacillus subtilus
- Eukaryotes: They can be a whole plant cell, animal cells
- Fungi cells like saccharomyces cervatiae.
The transfer of the recombinant vector (i.e. vector + Desired gene) is done such that the entire recombinant vector gets incorporated into the host cell genome.
The transfer is done by processes like
- Transformation: Here the host cell is subjected to minus 5-degree temp in presence of calcium chloride which acts as a desiccant to create sudden small cracks (openings) in the bacterial cell wall. During this cell wall cracks, the recombinant vector gets into the cell. Then by the sudden rise of temp. to 35 degrees (heat shock) the cell wall cracks are closed.
- Transduction: This is done by using a bacteriophage virus. Here the virus adheres to the bacterial cell wall and injects its genome into it.
- Conjugation: This a natural sexual process of bacteria exchanging their genome in the form of plasmid by forming inter-cell cytoplasmic bridges.
- and also by the use of liposomes, particle bombardment.
Multiplication and separation of cloned cells: The cloned cells can be separated by using antibiotics to kill non cloned cells.
The vectors used also have some antibiotic-resistant genes probes besides the desired gene in their genome. Hence when they enter the host cell, that host cell is resistant to specific antibiotics.
The cloned cells resist antibiotics and grow in large numbers. Or if the genetically modified organisms are used (Transgenic animals), they are reared to produce the desired product.
Expression of the desired gene inside the host cell:
Now that desired gene for insulin has got into the genome into E.Coli. But E.coli may not synthesize and produce insulin. Because E.Coli doesn’t require insulin for itself So there is no guarantee that E.Coli expresses that gene and produces insulin.
For this problem, we use gene expression agents like Lac operon or Tryptophan operon.
We will not go into details of them here. But in the presence of lactose in culture media, the lac-operon gene is active and there is the production of insulin by E.Coli and in the absence of lactose, there is no production of insulin.
For more details refer to Steps in genetic engineering
Applications of recombinant DNA technology varies for different sectors:
So let’s see the uses in different fields like
1. Recombinant DNA technology uses in medicine
- For production of vitamins like B12.
- For the production of antibiotics on large-scale.
- Recombinant proteins like insulin, other enzymes, hormones can be produced by rDNA technology.
- rDNA technology is used to produce recombinant vaccines in large-scale. Ex. Hepatitis-B vaccine
2. Recombinant DNA technology uses in animal husbandry and sericulture.
- For enhancement of milk production in cattle.
- For better meat yield in animals like pigs, cattle, birds.
- For enhancement of silk production in sericulture.
- For better egg yield in poultry birds.
- For better wool yield from sheep.
3. Recombinant DNA technology uses in agriculture.
- For biotechnology crops like cotton, vegetables, etc. rDNA technology can help produce high yielding plants with desired quality.
- Disease resistant crops like Bt-cotton, BT-brinjal are produced to withstand pest attack and thereby limit pesticide usage.
- For more, you may go through principles and applications of rDNA technology.