Birth of R-DNA
- Recombinant DNA is a form of DNA constructed in the laboratory. It is generated by transferring selected pieces of DNA from one organism to another.
- By 1970s we had understood the structure of DNA and central dogma. However, this was only theoretical. We could not separate a part of DNA which was coding for protein. In short, we did not know how to get access to DNA in order to change it.
- Incidentally in 1970s we understood how viruses work. Viruses get into the host cell and convert it into virus-producing factories.
- To do this they get into the host cell nucleus, insert their genome in the genome of host cell.
- The discovery of the action of a virus, that was affecting bacteria, led to development of R-DNA technology.
- In order to do this the virus (bacteriophage) had to do the following
- Identify a place to cut the bacterial genome
- Cut it
- Paste itself
- Make the new genome express itself in the bacterial cell.
- If we copied what the virus was doing, we have a way to ‘stich’ together DNA from 2 different sources. This is how R-DNA was born.
R-DNA technology: Manipulating the code of life
The basic steps in R-DNA are as follows:
- Identify the organism that has the gene of interest (one that expresses for desired trait, say production of Vitamin A in daffodil)
- Identify the target organism (To fortify rice with Vit A to make it Golden Rice)
- Take the organism that has the desired trait and cut it into pieces like a shredder. (daffodil genome)
- You can use enzymes restriction enzymes to cut a genome. (but this is cutting blindly, we did not know where we were cutting)
- Insert these pieces into a vector (vehicle to carry the genome to target organism)
- Find the vector with gene of interest (this is like finding the needle in a haystack, very tedious)
- Take the vector with gene of interest and multiply into colonies of the vector (gene that expresses for Vit A): This is called C-DNA library or gene library.
- Take the colony of vector with gene of interest and insert it in rice genome. (Pasting) This is done by an enzyme called DNA integrase (similar to what virus was doing).
- The resultant rice with gene from daffodil will be a recombinant DNA.
- This is how golden rice is made using R-DNA technology.
Vectors used in R-DNA technology
- Plasmid: Small circular pieces of DNA that is capable of replicating.
- Bacterial use plasmids to transfer anti-biotic resistance to the same generation.
- independently of chromosomal DNA of bacteria and that they could transfer genetic information.
- It was this process that gave host bacteria the capacity to inherit new genes and therefore new functions such as resistance to antibiotics.
- Alternately we can use bacteriophages (virus that infects bacteria), artificial chromosomes of yeast and bacteria as vectors in r-DNA technology.
Disadvantage of R-DNA
- R-DNA depends on finding the gene of interest based on the trait expressed in an organism.
- We had no way to pin point at the gene of interest. (we only know daffodil had Vit A producing gene, we dint know where exactly it was)
- We had cut the entire genome into pieces and finding one piece among millions of pieces.
- Thus it is very inefficient like finding a needle in a haystack.
Scope of R-DNA technology
R-DNA and Health
- Human insulin, was the 1st therapeutic proteins to be genetically cloned in E.coli using R-DNA technology
- Mono-clonal antibodies are made using R-DNA technology
- Production of vaccines against Hepatitis B
- Backbone of hepatitis and HIV diagnostic tests.
- Protein therapies such as human insulin, interferon and human growth hormone.
- Produce clotting factors for treating haemophilia.