Answer:
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Recombinant DNA (rDNA) = Vector DNA + Passenger DNA
In genetic engineering, a foreign gene is inserted into an organism's genome to alter its phenotype or to get useful products beneficial to humans.
STEP 1
In order to get the DNA, which is enclosed within the cell membranes, we have to break open the cell to release the DNA. For this, enzymes are used such as lysozyme (for bacteria), chitinase (for fungus) and cellulase (for plant cells).
This results in the release of DNA along with the other macromolecules like RNA, proteins (histones).
Now, in order to purify and separate the DNA, ribonuclease and protease are added to digest the RNA and Proteins to separate the DNA.
Next, the separated DNA is precipitated in the suspension by adding chilled ethanol and then it is separated out by spooling or winding over a fine appliance.
STEP 2
Now we have obtained the DNA. Next step is to cut the DNA by using the Restriction enzyme to get the desirable genes. The same restriction enzyme is used for both the vector DNA and the passenger DNA to produce the complementary cut parts (sticky ends). The cut out DNA fragments can be amplified to billions of times by PCR (Polymerase Chain Reaction). Agarose Gel Electrophoresis is done to separate the DNA fragments.
Vector DNA is ligated with Passenger DNA by the enzyme ligase to form the rDNA.
STEP 3
Now this rDNA is inserted into the bacterial cells by gene gun or bolistic gun, or by making them competent to take up the rDNA by treating them with divalent cations.
STEP 4
Bacterials cells are cultured in large containers called BioReactors where large amount of culture (100-1000 litres) can be processed at the same time.
STEP 5
Finally the product is obtained, purified and separated called Downstreaming Process.
The product is also formulated with the preservatives before marketing.
Example:- In 1983, Eli Lilly, an American Company produced the Genetically Engineered Insulin called 'Humulin'
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