Recombination DNA technology or rdna technology is a sophisticated molecular biology developed to produce essential biologicals on wide scale to match the demands of the huge population.
Benefits of recombinant dna technology are mind blowing, for instance the insulin required as 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 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 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 a 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 Human gene (DNA molecule) coding for insulin is isolated and transferred it to E.coli bacteria. Then the cloned bacteria is cultured in suitable media and grown on large-scale. This leads to profuse production of human insulin in large quantities.
See the video for a brief over view of rdna technology.
Principles of recombinant dna technology:
Recombination technology has following steps
Generation and isolation of desired gene.
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 desired gene: The desired gene is isolated from whole genome of the organism. For this two enzymes namely “Restriction endonucleases” and “DNA ligases” are used.
Endonucleases are the restriction enzymes which cut the DNA at specific points on the gene so as to get 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 molceules 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 are to be inserted into the genome of 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 host cell genome.
These vectors are large pieces of DNA molecules mostly. Plasmid is a circular, single stranded and self replicable DNA molecule present inside bacteria. They help in sexual reproduction of bacteria by transfer of genetic matter from one to another. Here we use them to transfer desired gene.
While bacteriophage is a virus which attacks bacteria and inserts its gene into bacterial cell for multiplication. Cosmid is similar to plasmid dna but can accommodate large DNA pieces.
This insertion of 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 desired gene) are transferred into host cells.
The host cells suitable for this purpose are
Prokaryotes: Bacteria like E.coli & Bacillus substillus
Eukaryotes: They can be whole plant cell, animal cells
Fungi cells like saccharomyces cervatiae.
The transfer of 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 are subjected to minus 5 degree temp in presence of calcium chloride which acts as 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 sudden rise of temp. to 35 degree (heat shock) the cell wall cracks are closed.
Transduction: This is done by using bacteriophage virus. Here the virus adhere to bacterial cell wall and injects it’s 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 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 desired gene in their genome. Hence when they enter host cell, that host cell is resistant to specific antibiotic.
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 inside the host cell:
Now that desired gene for insulin has got into 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 expressors like Lac operon or Tryptophan operon.
We will not go into details of them here. But in presence of lactose in culture media, the lac-operon gene is active and there is production of insulin by E.Coli and in the absence of lactose there is no production of insulin.
Applications of recombinant dna technology varies for different sectors:
So lets see the uses in different fields like
1. Recombinant dna technology uses in medicine
For production of vitamins like B12.
For 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 enhancement of silk production in seri culture.
For better meat yield in animals like pigs, cattle, birds.
For better egg yield.
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 with stand pest attack and there by limit pesticide usage.