Chapter 11 — Biotechnology: Principles and Processes — covers rDNA technology, tools (restriction enzymes, vectors, host organisms), and processes (PCR, gel electrophoresis, gene cloning). Carries 6-8 marks.
Key Concepts
Principles of Biotechnology
- Genetic Engineering: Altering DNA/RNA to modify an organism → recombinant DNA (rDNA) technology
- Bioprocess Engineering: Maintaining sterile conditions for large-scale production (bioreactors)
Tools of rDNA Technology
Restriction Enzymes (“Molecular Scissors”)
Cut DNA at specific palindromic recognition sequences
Example: EcoRI recognises 5′-GAATTC-3′ / 3′-CTTAAG-5′
Cuts between G and A → creates sticky ends (overhanging single-stranded regions)
Sticky ends are useful because they can join with complementary sticky ends from another DNA molecule using DNA ligase
Example: EcoRI recognises 5′-GAATTC-3′ / 3′-CTTAAG-5′
Cuts between G and A → creates sticky ends (overhanging single-stranded regions)
Sticky ends are useful because they can join with complementary sticky ends from another DNA molecule using DNA ligase
Vectors (Vehicles for Gene Transfer)
| Vector | Features |
|---|---|
| pBR322 (plasmid) | Origin of replication (ori), ampᴿ and tetᴿ genes (selectable markers), restriction sites |
| Ti plasmid | From Agrobacterium tumefaciens; T-DNA integrates into plant genome |
| Bacteriophage λ | Higher capacity than plasmid |
| BAC, YAC | For large DNA inserts (genomic libraries) |
Features of an ideal vector: (1) Origin of replication (ori), (2) Selectable marker (antibiotic resistance), (3) Cloning site (restriction enzyme sites), (4) Small size for easy manipulation.
Selectable Markers — Insertional Inactivation
If foreign gene is inserted into a selectable marker gene (e.g., ampᴿ), that gene is inactivated. Recombinants can be identified because they lose that antibiotic resistance.
Newer method: Blue-white screening using lacZ gene and X-gal — recombinants are white (lacZ disrupted), non-recombinants are blue.
Processes of rDNA Technology
PCR (Polymerase Chain Reaction)
Amplifies a specific DNA segment in vitro
Steps (each cycle):
1. Denaturation (94°C): DNA strands separate
2. Annealing (55-65°C): Primers bind to template
3. Extension (72°C): Taq polymerase extends primers → new DNA
After n cycles: 2ⁿ copies of target DNA
Taq polymerase: Thermostable DNA polymerase from Thermus aquaticus (works at high temp)
Steps (each cycle):
1. Denaturation (94°C): DNA strands separate
2. Annealing (55-65°C): Primers bind to template
3. Extension (72°C): Taq polymerase extends primers → new DNA
After n cycles: 2ⁿ copies of target DNA
Taq polymerase: Thermostable DNA polymerase from Thermus aquaticus (works at high temp)
Gel Electrophoresis
- Separates DNA fragments by size in agarose gel
- DNA is negatively charged → moves towards anode (+)
- Smaller fragments move faster/farther
- Visualised using ethidium bromide (stains DNA → orange under UV)
- Separated DNA cut from gel = elution
Gene Cloning Steps
1. Isolation of DNA (gene of interest)
2. Cutting with restriction enzymes (same enzyme for gene and vector)
3. Ligation (gene + vector → rDNA using DNA ligase)
4. Transfer into host cell (transformation, microinjection, biolistics/gene gun)
5. Selection of recombinants (selectable markers)
6. Expression of gene → desired protein
2. Cutting with restriction enzymes (same enzyme for gene and vector)
3. Ligation (gene + vector → rDNA using DNA ligase)
4. Transfer into host cell (transformation, microinjection, biolistics/gene gun)
5. Selection of recombinants (selectable markers)
6. Expression of gene → desired protein
Bioreactors
- Large vessels (100-1000L) for growing host cells at industrial scale
- Stirred-tank bioreactor: most common; agitator for mixing, O₂ supply, temperature/pH control
- Downstream processing: separation, purification of protein product
Quick Revision Points
- Restriction enzymes: cut at palindromic sequences → sticky ends; EcoRI (GAATTC)
- Vectors: pBR322 (ori, ampᴿ, tetᴿ); Ti plasmid (Agrobacterium, for plants)
- Selectable markers identify recombinants (insertional inactivation, blue-white screening)
- PCR: denature → anneal → extend; Taq polymerase; 2ⁿ copies
- Gel electrophoresis: DNA moves to +ve electrode; smaller = faster
- Gene cloning: isolate → cut → ligate → transform → select → express
- Bioreactors: stirred-tank; downstream processing for purification
Chapter Navigation
Previous: Microbes in Human Welfare Class 12 Notes
Next: Biotechnology and its Applications Class 12 Notes
Related Chapters in Class 12 Biology
- Biotechnology and its Applications Class 12 Notes
- Microbes in Human Welfare Class 12 Notes
- Human Health and Disease Class 12 Notes
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