Chapter 14 — Biotechnology
Key Concepts Quick Recap
| Term | Definition |
|---|
| Biotechnology | Use of living organisms or their components to develop useful products/processes |
| Recombinant DNA (rDNA) | DNA produced by joining DNA from different sources |
| Restriction endonuclease | Enzyme that cuts DNA at specific recognition sequences ("molecular scissors") |
| DNA ligase | Enzyme that joins (ligates) DNA fragments ("molecular glue") |
| Vector | DNA molecule used to carry foreign DNA into host cell (plasmid, phage) |
| Plasmid | Circular extrachromosomal DNA in bacteria — used as vector |
| PCR | Polymerase Chain Reaction — amplify specific DNA sequences in vitro |
| Gel electrophoresis | Separation of DNA fragments by size using electric field through agarose gel |
| Transgenic organism | Organism whose genome contains an introduced foreign gene |
| Tissue culture | In vitro growth of plant cells/tissues on artificial nutrient medium |
| Totipotency | Ability of a single plant cell to regenerate into a whole plant |
I. Choose the Best Answer (MCQs)
1. Restriction endonucleases are used in biotechnology to: (a) Join DNA fragments (b) Cut DNA at specific sequences (c) Amplify DNA (d) Separate DNA by size
Answer(b) Cut DNA at specific sequences Restriction endonucleases recognise specific short DNA sequences (4-8 bp palindromes) and cut both strands. Named for bacterium of origin: EcoRI (from E. coli), HindIII (from Haemophilus influenzae). They create sticky ends (staggered cuts) or blunt ends.
2. The backbone of recombinant DNA technology is: (a) DNA polymerase (b) Restriction enzymes and DNA ligase (c) RNA polymerase (d) Reverse transcriptase
Answer(b) Restriction enzymes and DNA ligase Restriction enzymes cut DNA at specific sites (both the gene of interest and the vector). DNA ligase joins the complementary sticky ends → recombinant DNA. Together, they are the molecular scissors and glue of genetic engineering.
3. PCR stands for: (a) Protein Chain Reaction (b) Polymerase Chain Reaction (c) Polynucleotide Chain Reaction (d) Phosphate Chain Reaction
Answer(b) Polymerase Chain Reaction PCR (Mullis, 1983; Nobel Prize 1993) amplifies specific DNA sequences exponentially using: DNA template + primers (specific oligonucleotides) + Taq DNA polymerase (heat-stable) + dNTPs. 3 steps: Denaturation (94°C) → Annealing (50-65°C) → Extension (72°C). Each cycle doubles the target DNA.
4. In gel electrophoresis, smaller DNA fragments: (a) Move slower (b) Stay at origin (c) Move faster (toward positive pole) (d) Move toward the negative pole
Answer(c) Move faster (toward positive pole) DNA is negatively charged (phosphate groups) → moves toward positive electrode. In agarose gel, smaller fragments move faster (less resistance from gel matrix). Larger fragments move slower and stay near the wells. DNA visualised using ethidium bromide staining under UV light.
5. Bt toxin produced by Bacillus thuringiensis is used against: (a) Fungi (b) Viruses (c) Specific insect larvae (d) All insects equally
Answer(c) Specific insect larvae Bt toxin (Cry protein) is a protoxin — activated by alkaline pH of insect gut → binds to epithelial cells → creates pores → cell swells → insect dies. It is specific to certain insect orders (Lepidoptera: bollworm; Diptera: mosquito larvae) and non-toxic to mammals and birds.
6. Totipotency was first demonstrated by: (a) Frederick Steward (b) Murashige and Skoog (c) Roger Ames (d) Walter Cannon
Answer(a) Frederick Steward Frederick Steward (1958) demonstrated totipotency by successfully growing a complete carrot plant from isolated phloem parenchyma cells in liquid culture medium. This proved that every plant cell contains the full genetic information to develop into a whole plant.
7. Golden rice is enriched with: (a) Vitamin C (b) Vitamin D (c) Vitamin A (β-carotene) (d) Iron
Answer(c) Vitamin A (β-carotene) Golden rice is a transgenic rice that produces β-carotene (precursor to Vitamin A) in the endosperm — giving it a golden colour. Developed by Ingo Potrykus and Peter Beyer. Genes for β-carotene biosynthesis (phytoene synthase + carotene desaturase) were introduced. Aims to address Vitamin A deficiency in developing countries.
II. Short Answer Questions
Q1. Describe the tools of recombinant DNA technology.
1. Restriction endonucleases (Molecular scissors):
Answer
- Recognise and cut double-stranded DNA at specific palindromic sequences (e.g. EcoRI recognises GAATTC).
- Create sticky ends (staggered cuts) for easier ligation.
- Used to isolate the gene of interest from donor DNA and to cut the vector.
2. DNA ligase (Molecular glue):
Answer
- Joins two DNA fragments by forming phosphodiester bonds between the sticky ends.
- Used to insert the gene of interest into the vector.
3. Vectors:
Answer
- Carry the foreign gene into the host cell.
- Types: Plasmids (most common), bacteriophages, cosmids, BAC (Bacterial Artificial Chromosomes), YAC (Yeast Artificial Chromosomes).
- Must have: origin of replication, selectable marker (antibiotic resistance), cloning site (MCS — multiple cloning site).
4. Host organism:
Answer
- Usually E. coli or Agrobacterium tumefaciens (for plants).
- Host cell takes up recombinant DNA → replicates and expresses the foreign gene.
5. PCR:
Answer
- Amplify specific DNA sequences before cloning.
Q2. Explain PCR with its steps.
Answer
PCR (Polymerase Chain Reaction) amplifies a specific DNA sequence exponentially in vitro.
Components needed:
- DNA template (target sequence)
- Two primers (forward and reverse; ~20 bp each)
- Taq DNA polymerase (from Thermus aquaticus — heat stable)
- dNTPs (deoxynucleotide triphosphates)
- Buffer and Mg²⁺
Three steps (one cycle): 1. Denaturation (94-95°C, 30 sec): Heat separates double-stranded DNA into two single strands. 2. Annealing (50-65°C, 30 sec): Primers bind to complementary sequences on each template strand. 3. Extension (72°C, 1 min/kb): Taq polymerase synthesises new DNA from 3' end of primers.
ResultAfter n cycles = 2ⁿ copies. After 30 cycles = ~10⁹ copies from 1 original molecule.
Applications: Forensics (DNA fingerprinting), diagnosis (COVID-19 RT-PCR), research (cloning, sequencing), ancestry testing.
Q3. What is tissue culture? Explain its steps and applications.
Answer
Tissue culture is the in vitro growth of isolated plant cells, tissues, or organs on artificial nutrient medium under sterile conditions.
Based on: Totipotency — every plant cell has the complete genetic information to regenerate a whole plant.
Steps: 1. Explant selection: A small piece of plant (leaf, root, bud, embryo) is selected. 2. Sterilisation: Surface-sterilised with sodium hypochlorite or alcohol. 3. Culture medium: Murashige and Skoog (MS) medium — minerals + sucrose + vitamins + plant hormones (auxin/cytokinin ratio). 4. Callus formation: Explant dedifferentiates → unorganised mass of cells (callus). 5. Organogenesis/Somatic embryogenesis: Hormones adjusted → callus differentiates → shoots + roots → complete plant (plantlet). 6. Hardening (acclimatisation): Plantlet transferred to soil gradually.
Applications:
- Mass propagation of elite plants (orchids, Banana, Potato).
- Production of disease-free plants (virus-free through meristem culture).
- Genetic engineering (transgenic plant production).
- Conservation of endangered plant species.
- Secondary metabolite production (pharmaceuticals from plant cells).
- Somatic hybridisation (protoplast fusion).
Common Mistakes
- Confusing restriction enzymes (cut DNA) and ligase (join DNA).
- Saying PCR requires a cell — it is an in vitro (cell-free) technique.
- Forgetting the role of Agrobacterium tumefaciens as a vector for plant transformation.
- Confusing callus (undifferentiated cells) and embryoid (differentiated embryo-like structure).
Exam Checklist
- Restriction enzymes: molecular scissors; palindromic sequences; sticky/blunt ends.
- DNA ligase: molecular glue; joins phosphodiester bonds.
- PCR: 3 steps (denaturation/annealing/extension); Taq polymerase; exponential amplification.
- Bt cotton: Cry gene from B. thuringiensis; kills bollworm; approved 2002 in India.
- Golden rice: β-carotene (Vitamin A); transgenic rice.
- Tissue culture: totipotency + steps: explant → callus → plantlet.
- Gel electrophoresis: smaller fragments → faster migration toward +ve electrode.
