Explain briefly the various processes of recombinant DNA technology.

r-DNA technology involves several steps in sequence such as,
* Isolation of the genetic material (DNA).
* Cutting of DNA at specific locations.
* Separation and isolation of desired DNA fragments.
* Amplification of gene of interest using PCR.
* Insertion of isolated gene into a suitable vector.
* Insertion of recombinant DNA into the Host Cell.
* Selection of transformed host cells.
* Obtaining the foreign gene product and downstream processing.
Isolation of the Genetic material (DNA) :
* DNA should be isolated in pure form, without other macromolecules. Hence, cell wall can be broken by treating the bacterial cells/ plant cells/ animal tissue with enzymes such as lysozyme (bacteria), cellulase (plant cells), chitinase (fungus)
* DNA should be removed from its histones proteins and RNA. This can be achieved by using enzymes ribonuclease for RNA and proteases for histone proteins.
Finally purified DNA precipitates out after the addition of child Ethanol.
Cutting of DNA at specific Locations :
* Restriction enzyme digestions are performed by incubating purified DNA molecules with the restriction enzyme. This process is also applicable to vector.
Separation and isolation of desired DNA fragments :
* DNA fragments can be separate using Agarose gel electrophoresis.
* DNA is a negatively charged molecule, hence it moves towards the positive electrode (anode) and are separated based on size.
* The separated bands of DNA are cut out from the Agarose gel and extracted from the gel piece called elution.
Amplification of Gene of Interest using PCR :


* PCR stands for polymerase chain Reaction.
* In this reaction, multiple copies of the gene of interest is synthesized in vitro using two sets of primers and the enzyme DNA polymerase (Taq polymerase).
* The process of replication of DNA is repeated many times, the segment of DNA can be amplified to approximately billion times.
* The amplified fragment if desired can now be used to ligate with a vector for further cloning.
Insertion of isolated gene into a suitable vector :
* The cut out gene of interest from the source DNA and the cut vector with space are mixed and ligase is added.
* This results in the preparation of recombinant DNA.
Insertion of Recombinant DNA into the Host cell/organism :
* There are several ways of introducing the ligated DNA into recipient cells.
* E.g. : Heat shock method - into bacterial cells.
* Microinjection - into animal cells.
* Gene gun method - into plant cells.
* Disarmed pathogen vectors - into plant and animal cells.
Selection of Transformed host cells :
* If a recombinant DNA bearing gene for resistance to an antibiotic (E.g.: ampicillin), if we spread the transformed cells on agar plates contain ampicillin, only transformants will grow, untransformed recipient cells will die. The ampicillin resistance gene in this case is called a selectable marker.
Obtaining the foreign gene product and downstream processing :
* The cells harbouring cloned genes of interest may be grown on a small scale in the laboratory. Large scale production is carried out in fermentors.
* The cultures may be used for extracting the desired proten and then purifying it by using different separation techniques.
* The processes include separation and purification, which are collectively referred to as downstream processing. Strict quality control testing for each product is also required.