Chapter 11: Biotechnology: Principles and Processes

Q1. What are the steps involved in recombinant DNA technology?

Solution

1. Isolation of desired gene
2. Cutting DNA with restriction enzymes
3. Joining DNA with ligase
4. Insertion into vector
5. Transfer into host cell
6. Selection and screening
7. Expression of desired gene

Q2. What is PCR? Name its three steps.

Solution

Polymerase Chain Reaction (PCR) amplifies specific DNA sequences.

Step 1 - Denaturation: 94-96°C (separate DNA strands)
Step 2 - Annealing: 55-65°C (primers bind)
Step 3 - Extension: 72°C (Taq polymerase extends primers)

Q3. What are restriction enzymes? How are they named?

Solution

Naming: Genus initial + species initials + strain + number
E.g., EcoRI = Escherichia coli RY13, first enzyme isolated

Q4. What are the types of vectors used in genetic engineering?

Solution

1. Plasmids (most common)
2. Bacteriophages (lambda phage)
3. Cosmids (hybrid of plasmid and phage)
4. BACs (Bacterial Artificial Chromosomes)
5. YACs (Yeast Artificial Chromosomes)

Q5. What is gel electrophoresis?

Solution

Process: (1) DNA loaded into agarose gel wells (2) Electric current applied (3) Smaller fragments move faster (4) DNA visualized using ethidium bromide and UV light (5) Bands cut for further use

Q6. What are the steps in downstream processing?

Solution

Steps: (1) Cell separation (2) Extraction (3) Purification (chromatography) (4) Formulation
Methods: Centrifugation, Filtration, Chromatography, Lyophilization

Q7. What is transformation? How is it achieved?

Solution

Methods: (1) Heat shock (2) CaCl₂ (3) Electroporation (4) Microinjection (5) Gene gun (6) Lipofection
Selectable markers help identify transformed cells

Q8. What is the role of bioreactors?

Solution

Types: (1) Stirred tank bioreactor (2) Bubble column bioreactor
Functions: (1) Maintains optimal conditions (pH, temperature, O₂) (2) Aeration and mixing (3) Nutrient supply (4) Large-scale production

Q9. What is the difference between upstream and downstream processing?

Solution

Upstream: Steps before fermentation - cell culture, media preparation
Downstream: Steps after fermentation - separation, purification, formulation

Q10. What are blunt ends and sticky ends?

Solution

Blunt ends: Both strands cut at same position (e.g., HaeIII)
Sticky ends: Staggered cuts create overhangs (e.g., EcoRI)
Sticky ends useful for joining DNA from different sources

Q11. What are the properties of a good cloning vector?

Solution

1. Origin of replication (ori)
2. Selectable marker (antibiotic resistance)
3. Unique restriction sites (MCS)
4. Small size
5. High copy number
6. Ability to host large DNA inserts

Q12. What is the difference between cloning and expression vectors?

Solution

Cloning: Replicate DNA (e.g., pUC19, pBR322)
Expression: Express gene into protein (e.g., pET, pGEX)

Q13. What is the principle of PCR amplification?

Solution

After n cycles: 2ⁿ copies of target DNA
30 cycles = ~1 billion copies (2³⁰)

Q14. What is the function of DNA ligase?

Solution

Uses: (1) Joining DNA fragments after restriction digestion (2) Sealing Okazaki fragments (3) Joining insert DNA with vector

Q15. What is the difference between prokaryotic and eukaryotic expression systems?

Solution

Prokaryotic (E. coli): Fast, cheap, no post-translational modification
Eukaryotic (Yeast, CHO): Slower, proper folding, glycosylation, but complex

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