Processes of Recombinant DNA Technology (NEET aspect)

Описание: Recombinant DNA technology involves altering genetic material by introducing a foreign DNA into a host organism.

1. Isolation of Genetic Material (DNA)
DNA is isolated from:
Donor organism (gene of interest)
Host organism (usually plasmid DNA)
DNA is released by breaking the cell using:
Enzymes like lysozyme (bacteria)
Cellulase (plants)
DNA is purified by removing proteins and RNA.

2. Cutting of DNA at Specific Locations
Done using restriction endonucleases
These enzymes cut DNA at specific palindromic sequences
Produces:
Sticky ends or blunt ends
Same enzyme cuts both:
Foreign DNA
Vector DNA
ensures proper joining
Example: EcoRI

3. Amplification of Gene of Interest (PCR)
Gene is multiplied using Polymerase Chain Reaction (PCR)
Requires:
Template DNA
Primers
Taq polymerase
dNTPs
Steps:
Denaturation
Annealing
Extension

4. Ligation of DNA Fragment
The foreign DNA is joined to the vector using DNA ligase
Forms recombinant DNA
Sticky ends help in easy ligation

5. Insertion of Recombinant DNA into Host Cell (Transformation)
Recombinant DNA is introduced into host (commonly E. coli)
Methods:
Heat shock
Electroporation
Microinjection
Host cell takes up recombinant DNA

6. Selection and Screening of Recombinants
Only cells with recombinant DNA are selected
Methods include:
Antibiotic resistance markers
Blue-white screening
Non-recombinants are eliminated

7. Obtaining the Foreign Gene Product
Host cells are cultured on a large scale
Gene expresses desired protein
Product is extracted and purified

8. Downstream Processing
Separation and purification of product
Formulation into:
Medicines
Vaccines
Enzymes

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