🌸Chapter 9
Biotechnology: Principles and Processes
(5Marks)
Q1.
Explain the steps involved in the production of recombinant human insulin using
E. coli.
Ans:
1.
Human
insulin gene is isolated and inserted into a plasmid vector.
2.
Recombinant
plasmid introduced into E. coli (transformation).
3.
Bacteria
express A and B polypeptide chains separately.
4.
Chains
are purified and combined via disulfide bonds to produce functional insulin.
5.
The
insulin is tested and formulated for medical use.
Q2.
Describe the process of PCR and explain its applications in biotechnology.
Ans:
Process:
- Denaturation (95°C): DNA
strands separate.
- Annealing (55°C): Primers bind
to target DNA.
- Extension (72°C): Taq
polymerase synthesizes new DNA strands.
- Repeated cycles amplify the DNA
millions of times.
Applications:
- Diagnosis of genetic disorders
and infectious diseases
- Gene cloning
- DNA fingerprinting
- Detection of pathogens
Q3.
Explain how gel electrophoresis is used to separate DNA fragments.
Ans:
- DNA samples mixed with loading
dye are loaded into agarose gel wells.
- Electric current applied; DNA
moves toward the positive electrode due to negative charge.
- Smaller fragments move faster
than larger fragments.
- DNA visualized using ethidium
bromide under UV light.
- Band size compared with a DNA
ladder for identification.
Q4.
Explain the role of restriction enzymes in recombinant DNA technology. Give
examples.
Ans:
- Restriction enzymes cut DNA at
specific palindromic sequences.
- They produce sticky ends
or blunt ends for DNA ligation.
- Example: EcoRI (sticky end),
HindIII (sticky end), SmaI (blunt end).
- Essential for precise gene
cloning and recombinant DNA formation.
Q5.
Describe the structure and features of a typical plasmid vector used in genetic
engineering.
Ans:
- Circular, double-stranded DNA
- Contains origin of replication
(Ori)
- Selectable markers (e.g.,
amp^r, tet^r)
- Unique restriction sites
(multiple cloning sites)
- Small size for easy uptake by
host cells
- Example: pBR322
Q6.
Explain the process of bacterial transformation.
Ans:
1.
Bacterial
cells made competent using CaCl₂ or heat-shock.
2.
Recombinant
DNA introduced into the cells.
3.
Transformed
cells survive in selective medium (e.g., antibiotic-containing).
4.
Non-transformed
cells die.
5.
Transformed
cells multiply and express the desired gene.
Q7.
How can recombinant DNA molecules be introduced into eukaryotic cells?
Ans:
- Transfection: Introduction of DNA using
liposomes, electroporation, or microinjection.
- Gene gun/biolistics: DNA-coated particles shot into
plant cells.
- Agrobacterium-mediated
transformation:
Transfer of DNA into plant genomes.
- Viral vectors: Modified viruses deliver DNA
into eukaryotic cells.
Q8.
Explain the principle and steps of blue-white screening for recombinant
colonies.
Ans:
- Plasmid has lacZ gene
coding for β-galactosidase.
- Insertion of foreign DNA
disrupts lacZ (insertional inactivation).
- Colonies grown on X-gal medium:
- White colonies = recombinant
(foreign DNA inserted)
- Blue colonies =
non-recombinant (lacZ functional)
Q9.
What is a bioreactor? Explain its role in industrial biotechnology.
Ans:
- Large vessel providing
controlled conditions (pH, temperature, oxygen, nutrients)
- Supports growth of microbial or
mammalian cells for product formation
- Ensures large-scale production
of recombinant proteins, vaccines, and enzymes
Q10.
Describe the difference between stirred-tank and airlift bioreactors.
Ans:
|
Feature |
Stirred-Tank |
Airlift |
|
Mixing |
Mechanical impeller |
Air bubbles |
|
Shear stress |
High |
Low |
|
Applications |
Wide variety of cultures |
Shear-sensitive cultures |
- Both used in large-scale
industrial production.
Q11.
Explain downstream processing and its significance in biotechnology.
Ans:
- Involves purification,
separation, and formulation after fermentation.
Steps:
1.
Cell
removal (filtration/centrifugation)
2.
Protein
purification (chromatography, precipitation)
3.
Formulation
and stabilization
Significance:
- Ensures product purity and
safety for medical or industrial use.
Q12.
Discuss the applications of recombinant DNA technology in agriculture.
Ans:
- Production of pest-resistant
crops (Bt cotton)
- Herbicide-resistant crops
(Roundup Ready soybeans)
- Nutritionally enhanced crops
(Golden rice with β-carotene)
- Disease-resistant plants and
improved yield
Q13.
Explain how human growth hormone (hGH) is produced using biotechnology.
Ans:
1.
hGH
gene inserted into plasmid vector
2.
Recombinant
plasmid introduced into E. coli
3.
Bacteria
produce hGH protein
4.
Protein
purified and formulated
5.
Used
to treat growth hormone deficiencies
Q14.
How is DNA visualized after electrophoresis? Why is this important?
Ans:
- Stain agarose gel with ethidium
bromide
- DNA fluoresces under UV light
- DNA fragments can be sized and analyzed
- Important for confirming
successful cloning or PCR amplification
Q15.
Describe the principle of recombinant DNA technology.
Ans:
- DNA from different sources
combined to form a recombinant molecule.
- Introduced into a host for
replication or expression.
- Key tools: restriction enzymes,
ligase, vectors, competent cells.
Q16.
Explain the use of PCR in pathogen detection.
Ans:
- DNA from pathogen amplified
using specific primers.
- Millions of copies produced in
few hours.
- Allows rapid, sensitive detection
of bacteria/viruses (e.g., COVID-19, TB).
- Can detect small amounts of DNA
from clinical samples.
Q17.
Explain the role of Taq polymerase in PCR.
Ans:
- Heat-stable DNA polymerase from
Thermus aquaticus
- Synthesizes DNA at high
temperatures during extension
- Allows multiple PCR cycles
without enzyme denaturation
Q18.
How are sticky ends and blunt ends different? Why are sticky ends preferred?
Ans:
- Sticky ends: single-stranded
overhangs, easily annealed with complementary DNA
- Blunt ends: straight cuts,
harder to ligate
- Sticky ends are preferred for
high-efficiency ligation in recombinant DNA technology
Q19.
Explain the role of selectable markers in plasmid vectors.
Ans:
- Allow identification of cells
that have taken up the recombinant DNA
- Example: antibiotic resistance
genes (amp^r, tet^r)
- Non-transformed cells die in
selective medium
Q20.
Describe the steps involved in isolating DNA from a bacterial cell.
Ans:
1.
Cell
lysis using detergent or enzyme (lysozyme)
2.
Removal
of proteins and RNA using protease/RNase
3.
Precipitation
of DNA using cold ethanol
4.
Purified
DNA used for cloning or PCR
Q21.
Explain the structure and function of plasmids used in biotechnology.
Ans:
- Circular DNA molecule
- Ori: allows self-replication
- Selectable markers: identify
transformed cells
- Multiple cloning sites: insert
foreign DNA
- Function: vector to replicate
and express genes in host
Q22.
Discuss the advantages of recombinant DNA technology over traditional methods.
Ans:
- Faster and precise
- Can transfer genes across
species
- Produces large-scale pure
proteins
- Enables gene therapy, GM crops,
and molecular diagnostics
Q23.
How is recombinant DNA used in the production of vaccines?
Ans:
- Pathogen genes cloned into
vectors
- Expressed in host cells to
produce antigens
- Purified antigens used as
vaccines (e.g., Hepatitis B vaccine)
- Safe, effective, and
mass-producible
Q24.
Explain the process of transformation in detail.
Ans:
1.
Bacteria
made competent
2.
Recombinant
plasmid DNA introduced
3.
Cells
incubated for uptake and recovery
4.
Transformed
cells selected using antibiotics
5.
Positive
colonies grow and produce desired protein
Q25.
Describe the role of a bioreactor in large-scale protein production.
Ans:
- Provides optimal growth
conditions (pH, temperature, oxygen)
- Ensures homogenous mixing and
nutrient supply
- Facilitates high-density
culture of host cells
- Enables continuous or batch
production of proteins like insulin or enzymes
Q26.
Explain how a recombinant plasmid is constructed.
Ans:
1.
Vector
and foreign DNA cut with same restriction enzyme
2.
Sticky
ends anneal
3.
DNA
ligase seals nicks forming recombinant DNA
4.
Introduced
into host cell (transformation)
5.
Recombinant
colonies identified using markers
Q27.
How are recombinant DNA molecules screened?
Ans:
- Using antibiotic selection:
only transformed cells survive
- Using blue-white screening:
white colonies = recombinants
- Colony PCR or restriction
digestion can also confirm recombinants
Q28.
Explain the importance of palindromic sequences in DNA cloning.
Ans:
- Restriction enzymes recognize
palindromic sequences
- Cuts produce sticky ends for
ligation
- Ensures precise and
reproducible gene cloning
Q29.
Explain how recombinant DNA technology is used in agriculture.
Ans:
- Bt gene inserted into crops →
pest resistance
- Herbicide resistance genes →
weed control
- Golden rice with β-carotene →
enhanced nutrition
- Disease-resistant crops →
improved yield
Q30.
How does downstream processing ensure product quality?
Ans:
- Removes cells and impurities
- Purifies protein/enzyme via
chromatography
- Stabilizes final product
- Ensures safety, activity, and
efficacy
Q31.
Explain how recombinant DNA technology can produce industrial enzymes.
Ans:
- Genes coding for enzymes cloned
into microbial hosts
- Microbes cultured in
bioreactors
- Enzymes harvested, purified,
and used in detergents, food processing, or pharmaceuticals
Q32.
What are competent cells? How are they prepared?
Ans:
- Cells that can take up DNA
- Prepared by:
- Treating bacterial cells with
CaCl₂ at 0–4°C
- Heat-shock briefly to allow
DNA uptake
Q33.
Explain the role of ligase in gene cloning.
Ans:
- DNA ligase forms phosphodiester
bonds between vector and foreign DNA
- Seals nicks, producing stable
recombinant DNA
- Essential for successful gene
cloning
Q34.
How is recombinant DNA technology applied in medicine?
Ans:
- Production of insulin,
interferons, vaccines
- Gene therapy for genetic
disorders
- Molecular diagnostics and
pathogen detection
Q35.
Explain the principle of electrophoresis and its applications.
Ans:
- DNA moves toward positive
electrode due to negative charge
- Smaller fragments move faster
Applications: - Separation of DNA fragments
- Checking recombinant DNA
- DNA fingerprinting and
diagnostics
Q36.
Describe the use of multiple cloning sites in plasmid vectors.
Ans:
- Contains several unique
restriction sites
- Provides flexibility to insert
different DNA fragments
- Facilitates recombinant DNA
construction
Q37.
How is recombinant DNA technology used to produce growth hormone?
Ans:
- hGH gene inserted into plasmid
vector
- Expressed in E. coli
- Protein purified and used to
treat deficiencies
Q38.
Explain the steps of downstream processing in detail.
Ans:
1.
Cell
removal by centrifugation/filtration
2.
Concentration
of product
3.
Purification
via chromatography
4.
Formulation
and stabilization
5.
Quality
control tests
Q39.
Discuss the use of recombinant DNA technology in diagnostics.
Ans:
- PCR amplifies specific pathogen
DNA
- Rapid and sensitive detection
of infections
- Can detect genetic disorders
- Used in forensic studies (DNA
fingerprinting)
Q40.
Explain the differences between cloning vector and expression vector.
Ans:
|
Feature |
Cloning Vector |
Expression Vector |
|
Purpose |
Gene replication |
Gene expression as protein |
|
Elements |
Ori, selectable marker |
Ori, marker, promoter |
|
Host |
Bacteria |
Bacteria/Yeast |
Q41.
Describe how recombinant vaccines are produced.
Ans:
- Pathogen gene cloned into
vector
- Expressed in host cells to
produce antigen
- Purified antigen formulated as
vaccine
- Safe alternative to live-attenuated
vaccines
Q42.
Explain the significance of PCR in forensic science.
Ans:
- Amplifies minute DNA samples
- Enables DNA fingerprinting from
hair, blood, or tissue
- Identifies suspects or victims
in criminal investigations
Q43.
Explain how recombinant DNA technology improves crop yield.
Ans:
- Pest-resistant (Bt) crops →
reduced losses
- Herbicide-resistant crops →
easier weed control
- Nutritionally enhanced crops →
improved quality
- Disease-resistant crops →
higher survival rate
Q44.
Explain the steps in the production of a recombinant protein.
Ans:
1.
Isolation
of target gene
2.
Cloning
into vector
3.
Transformation
into host
4.
Culture
in bioreactor
5.
Purification
(downstream processing)
6.
Quality
testing and formulation
Q45.
How does Taq polymerase differ from DNA polymerase of other organisms?
Ans:
- Heat-stable enzyme from Thermus
aquaticus
- Functions at 72°C during PCR
extension
- Other polymerases denature at
high temperature
Q46.
Explain the role of selectable markers in recombinant DNA experiments.
Ans:
- Allows identification of
transformed cells
- Non-transformed cells die under
selective conditions
- Ensures only desired
recombinants survive
Q47.
How are recombinant proteins purified?
Ans:
- Cells removed by
centrifugation/filtration
- Chromatography techniques
separate target protein
- Precipitation, dialysis, or
affinity purification used
- Final product tested for purity
Q48.
Explain the importance of palindromic sequences in molecular cloning.
Ans:
- Restriction enzymes recognize
palindromic sequences
- Cuts generate sticky ends for
ligation
- Enables precise DNA
manipulation
Q49.
Discuss the applications of biotechnology in industry.
Ans:
- Enzyme production (amylase,
protease)
- Antibiotics and vaccines
- Biofuels (ethanol)
- Waste treatment using microbes
Q50.
Explain the advantages of recombinant DNA technology.
Ans:
- Produces pure, safe proteins
and medicines
- Cross-species gene transfer
possible
- Faster and more precise than
traditional methods
- Enables genetic modification of
crops, diagnostics, and therapeutics
