NEET Biology - Unit 9- Principles and process of Biotechnology- Study Notes - New Syllabus

Principles and Process of Biotechnology: Genetic Engineering (Recombinant DNA Technology)

📌 Definition of Biotechnology

According to the European Federation of Biotechnology:

“The integration of natural sciences and organisms, cells, parts thereof, and molecular analogues for products and services.”

Key idea: Using living systems or components of living systems to develop products and solve problems.

🌱 Principles of Biotechnology

Modern biotechnology is based on two main techniques:

• Genetic Engineering (Recombinant DNA Technology)
  • Alteration of DNA or RNA to introduce desired traits into host organisms.
  • Changes the phenotype of the host.
• Microbial/Chemical Engineering Principles
  • Maintaining sterile conditions for large-scale growth of microbes or eukaryotic cells.
  • Production of enzymes, vaccines, antibiotics, and other biotechnological products.

💡 Conceptual Background of Genetic Engineering

Sexual reproduction introduces variation, whereas asexual reproduction preserves genetic information.

This concept led to gene isolation, transfer, and cloning without introducing unwanted genes.

Example: 1972, Herbert Boyer and Stanley Cohen cut a plasmid DNA and isolated an antibiotic resistance gene using restriction enzymes and linked it to a plasmid via DNA ligase → first recombinant DNA molecule.

🛠 Tools of Recombinant DNA Technology

1. Restriction Enzymes

• Discovered in 1963 to protect bacteria from bacteriophages. 
• Recognize specific DNA sequences (recognition sites) and cut DNA at precise locations.
• Types:
  • Endonucleases: Cut within DNA strand.
  • Exonucleases: Remove nucleotides from DNA ends.
• Example: Hind II, EcoRI.
• Palindromic sequences: Same forward & backward.
  5’ – GAATTC – 3’
  3’ – CTTAAG – 5’
• Cutting leaves sticky ends → useful for joining DNA fragments.

2. Polymerase Enzymes

• Used in PCR to amplify DNA segments.
• Example: Taq polymerase from Thermus aquaticus is thermostable, used in high-temperature cycles of PCR.

3. Ligases

• Join DNA fragments by forming phosphodiester bonds.
• Essential for creating recombinant DNA.

4. Vectors

• DNA molecules that carry foreign genes into host cells.
• Examples: Plasmids, Bacteriophages, Viruses (e.g., Retrovirus, Agrobacterium T-DNA).
• Features of a good vector:
  • Origin of replication: Allows replication of inserted DNA.
  • Selectable marker: Distinguishes transformants from non-transformants.
  • Cloning sites: Restriction sites for inserting foreign DNA.

5. Host Organisms

• Bacteria (e.g., E. coli) most commonly used.
• Host cells must be made competent (e.g., Ca²⁺ treatment, microinjection in animal cells) to take up recombinant DNA.

🔬 Steps in Recombinant DNA Technology

Isolation of DNA

• Break cells → release DNA from nucleus, mitochondria, or plasmids.
• Enzymes used: cellulase (plants), lysozyme (bacteria), chitinase (fungi).

Cutting DNA with Restriction Enzymes

• Cuts DNA at specific sites → generates fragments with sticky ends.
• Vector DNA is cut with same enzyme for complementary ends.

Separation of DNA Fragments

• Gel Electrophoresis:
• DNA is negatively charged → moves towards anode.
• Agarose gel acts as a sieve → separates by size.
• Visualized by ethidium bromide under UV light.

Ligation of DNA

• DNA fragments joined with DNA ligase → recombinant DNA formed.

Insertion into Host Cells

• Methods:
  • Bacterial transformation (competent cells)
  • Microinjection in animal cells
  • Viral vectors (Agrobacterium in plants, retrovirus in animals)

Amplification of Gene (PCR)

• Repeated cycles of denaturation, annealing, extension using primers and Taq polymerase.
• Produces billions of copies of DNA in vitro.

Expression of Foreign Gene

• Recombinant DNA expressed → produces desired protein.
• Conditions optimized for promoter, codon usage, host strain.

Downstream Processing

• Separation, purification, and formulation of product.
• Quality control before market release.
• Example products: insulin, growth hormones, vaccines, enzymes.

💡 Summary Table: Recombinant DNA Technology

📝 Quick Recap 
Biotechnology: Using living organisms/components for products/services.
Principles: Genetic engineering + microbial/chemical engineering.
Genetic Engineering: Alter DNA → recombinant DNA → host → desired product.
Tools: Restriction enzymes, ligases, vectors, polymerases.
Vectors: Plasmids, bacteriophages, viruses; must have origin, selectable marker, cloning site.
Host Cells: Bacteria, plant, animal; made competent for DNA uptake.
PCR: Amplifies DNA using primers & Taq polymerase.
Bioreactors: Large vessels to produce recombinant proteins under controlled conditions.
Products: Insulin, growth hormones, vaccines, enzymes, antibiotics.