Biotechnology: Principles and Processes

NCERT Line-by-Line Breakdown for NEET 2026

Unit 4: Biotechnology

Biotechnology deals with techniques of using live organisms or enzymes from organisms to produce products and processes useful to humans. The European Federation of Biotechnology (EFB) defines it as: “The integration of natural science and organisms, cells, parts thereof, and molecular analogues for products and services.”

1. Principles of Biotechnology

1. Genetic Engineering
Techniques to alter the chemistry of genetic material (DNA/RNA), to introduce these into host organisms and thus change the phenotype of the host organism.

2. Bioprocess Engineering
Maintenance of sterile (microbial contamination-free) ambience to enable growth of only the desired microbe/eukaryotic cell in large quantities for products like antibiotics, vaccines, enzymes, etc.

First Recombinant DNA

Constructed by Stanley Cohen and Herbert Boyer (1972). They isolated the antibiotic resistance gene from a plasmid of Salmonella typhimurium and linked it with a plasmid vector.

2. Tools of Recombinant DNA Technology

A. Restriction Enzymes (Molecular Scissors)

Belong to nucleases class. Two types: Exonucleases (remove nucleotides from ends) and Endonucleases (cut at specific positions within DNA).

First Discovered: Hind II (Cuts at a specific sequence of 6 base pairs).
Recognition Sequence: Palindromic Nucleotide Sequences (Read same forward and backward, e.g., MALAYALAM).
Action: Cuts sugar-phosphate backbone a little away from the center of palindrome. produces Sticky Ends (facilitate joining by DNA Ligase).

Naming Convention (EcoRI):
E = Genus (Escherichia); co = Species (coli); R = Strain (RY13); I = Order of isolation.

B. Separation of DNA Fragments

Gel Electrophoresis: DNA is negatively charged, so it moves towards Anode (+) through a matrix (Agarose gel).

Smaller fragments move farther.
Visualization: Staining with Ethidium Bromide followed by exposure to UV radiation → Bright Orange Bands.
Elution: Extraction of DNA band from the gel piece.

C. Cloning Vectors (Vehicles)

Plasmids and Bacteriophages. Example: pBR322 (E. coli cloning vector).

Feature	Function
Ori (Origin of Replication)	Sequence where replication starts. Controls Copy Number.
Selectable Marker	Helps identify and eliminate non-transformants. (e.g., Ampicillin resistance gene amp^R, Tetracycline resistance gene tet^R).
Cloning Sites	Recognition sites for restriction enzymes (e.g., BamHI, SalI).

🧬 Insertional Inactivation (Blue-White Screening):
Instead of antibiotics, a chromogenic substrate is used.
• Recombinant bacteria: Insert inactivates β-galactosidase gene → No color (White colonies).
• Non-recombinant bacteria: Enzyme active → Blue colonies.

D. Competent Host

DNA is hydrophilic, cannot pass through membrane. Methods to force entry:

Chemical: Treating with divalent cation (Calcium) + Heat Shock (42°C).
Micro-injection: Recombinant DNA directly injected into nucleus (Animal cells).
Biolistics / Gene Gun: Gold/Tungsten particles coated with DNA bombarded (Plant cells).
Disarmed Pathogen: Agrobacterium tumefaciens (Ti plasmid) for plants; Retroviruses for animals.

3. Processes of Recombinant DNA Technology

A. Isolation of Genetic Material (DNA)

Cell wall broken by enzymes: Lysozyme (bacteria), Cellulase (plants), Chitinase (fungi). RNA removed by Ribonuclease, Proteins by Protease. Chilled Ethanol precipitates purified DNA (seen as fine threads – Spooling).

B. Amplification using PCR

Polymerase Chain Reaction (Kary Mullis). Amplifies gene of interest billion times.

Denaturation (94°C): dsDNA separates into ssDNA.
Annealing (50-60°C): Primers bind to complementary regions.
Extension (72°C): Taq Polymerase (thermostable enzyme from Thermus aquaticus) adds nucleotides.

C. Bioreactors (Large Scale Production)

Vessels (100-1000 Liters) where raw materials are biologically converted into specific products using microbial/plant/animal cells. Provides optimal growth conditions (Temp, pH, substrate, salts, vitamins, oxygen).

Stirred-tank reactor: Most common. Has agitator system for mixing and oxygen availability.
Sparged stirred-tank reactor: Air bubbles increase surface area for oxygen transfer.

D. Downstream Processing

The processes after the biosynthetic stage:
1. Separation
2. Purification
Product is formulated with preservatives and undergoes quality control testing.

📝 Rapid Fire MCQs

Q1. Which of the following is a palindromic sequence?

A) 5′-CGTATG-3′
B) 5′-CGAATG-3′
C) 5′-GAATTC-3′
D) 5′-GACTAC-3′

Click to check Answer

Answer: C) 5′-GAATTC-3′ (Reads 3′-CTTAAG-5′ on opposite strand – EcoRI site).

Q2. The DNA polymerase used in PCR is isolated from:

A) Escherichia coli
B) Agrobacterium tumefaciens
C) Thermus aquaticus
D) Salmonella typhimurium