🌸Chapter 9
Biotechnology: Principles and Processes
(4Marks)
Q1.
Explain the difference between genetic engineering and traditional breeding.
Ans:
- Genetic engineering: Direct manipulation of DNA to
produce desired traits, can transfer genes across species, fast and
precise.
- Traditional breeding: Involves crossing of organisms
with desirable traits, limited to closely related species, slow and less
precise.
Q2.
List the steps involved in recombinant DNA technology.
Ans:
1.
Isolation
of DNA from donor organism
2.
Cutting
DNA using restriction enzymes
3.
Ligation
of foreign DNA into a vector
4.
Introduction
of recombinant DNA into host (transformation)
5.
Screening
and selection of recombinants
6.
Large-scale
culture and downstream processing
Q3.
Explain the role of restriction enzymes in biotechnology.
Ans:
- Cut DNA at specific palindromic
sequences
- Produce sticky or blunt ends
for gene insertion
- Essential for precise gene
cloning and recombinant DNA construction
Q4.
What are plasmids? How are they used in gene cloning?
Ans:
- Plasmids are small circular DNA
molecules in bacteria, independent of chromosomal DNA.
- Used as vectors to carry
foreign DNA into host cells for replication and expression.
- Contain selectable markers
(e.g., amp^r) and unique restriction sites for cloning.
Q5.
Differentiate between sticky ends and blunt ends.
Ans:
|
Feature |
Sticky Ends |
Blunt Ends |
|
Structure |
Single-stranded overhangs |
No overhang, straight cut |
|
Ligation |
Easily ligated with complementary ends |
Harder to ligate |
|
Formation |
Some restriction enzymes (EcoRI) |
Some restriction enzymes (SmaI) |
Q6.
Explain the role of DNA ligase.
Ans:
- DNA ligase joins DNA fragments
by forming phosphodiester bonds.
- Seals nicks in sugar-phosphate
backbone of recombinant DNA.
- Essential for creating stable
recombinant DNA molecules.
Q7.
Describe the structure and important features of pBR322.
Ans:
- Circular plasmid vector
- Contains origin of replication
(Ori)
- Selectable markers: ampicillin
(amp^r) and tetracycline (tet^r)
- Unique restriction sites for
inserting foreign DNA
- Small size facilitates
transformation into E. coli
Q8.
Explain the process of bacterial transformation.
Ans:
1.
Bacteria
made competent using CaCl₂ or heat-shock
2.
Recombinant
plasmid DNA introduced into bacterial cell
3.
Transformed
cells selected using antibiotic resistance
4.
Successfully
transformed bacteria replicate and express foreign gene
Q9.
What is PCR? Explain its significance.
Ans:
- PCR (Polymerase Chain Reaction) amplifies a specific DNA
fragment in vitro.
- Steps: Denaturation, Annealing,
Extension.
- Uses Taq polymerase from Thermus
aquaticus.
- Significant for gene cloning,
diagnostics, DNA fingerprinting, pathogen detection.
Q10.
Explain the three steps of PCR.
Ans:
1.
Denaturation
(95°C): DNA
strands separate
2.
Annealing
(55°C): Primers
bind to complementary DNA sequences
3.
Extension
(72°C): Taq
polymerase synthesizes new DNA strands
- Repeated cycles yield millions
of copies of target DNA.
Q11.
How is recombinant insulin produced using E. coli?
Ans:
1.
Human
insulin gene inserted into plasmid vector.
2.
Plasmid
introduced into E. coli (transformation).
3.
E.
coli expresses A and B chains
separately.
4.
Chains
purified and combined to form functional insulin.
Q12.
What is an expression vector? Give one example.
Ans:
- A vector designed to express
the inserted gene in the host organism.
- Contains promoter, cloning
site, and selectable marker.
- Example: pET vector for
protein expression in E. coli.
Q13.
Describe the principle and steps of gel electrophoresis.
Ans:
- Principle: DNA fragments are negatively
charged and move toward the anode through agarose gel; smaller fragments
move faster.
- Steps:
1.
DNA
samples mixed with loading dye
2.
Loaded
into agarose gel wells
3.
Electric
current applied
4.
DNA
visualized using ethidium bromide under UV light
Q14.
What is the function of a bioreactor?
Ans:
- Provides controlled conditions
(pH, temperature, oxygen, nutrients)
- Supports large-scale microbial
or cell culture
- Facilitates continuous
production of recombinant proteins, enzymes, vaccines
Q15.
Differentiate between stirred-tank and airlift bioreactors.
Ans:
|
Feature |
Stirred-Tank |
Airlift |
|
Mixing |
Mechanical impeller |
Air bubbles |
|
Shear |
High |
Low |
|
Applications |
Wide variety of cultures |
Shear-sensitive cultures |
Q16.
What is downstream processing in biotechnology?
Ans:
- Purification, separation, and
formulation of the desired product after fermentation.
- Steps include: cell removal,
filtration, chromatography, and quality testing.
Q17.
Explain the role of sparger and agitator in a bioreactor.
Ans:
- Sparger: Introduces sterile air/oxygen
for aerobic growth
- Agitator: Ensures uniform mixing,
prevents sedimentation, distributes nutrients
Q18.
Define selectable marker and give examples.
Ans:
- Gene used to identify transformed
cells
- Example: ampicillin resistance
(amp^r), tetracycline resistance (tet^r)
Q19.
Explain insertional inactivation.
Ans:
- A foreign DNA inserted into a
marker gene (e.g., lacZ) inactivates it.
- Recombinant colonies can be
distinguished (white colonies) from non-recombinants (blue colonies) using
X-gal.
Q20.
What are competent cells and how are they prepared?
Ans:
- Cells that can take up foreign
DNA
- Prepared using CaCl₂
treatment or heat-shock to make membrane permeable
Q21.
Explain the difference between transformation and transfection.
Ans:
- Transformation: Uptake of naked DNA by
bacteria
- Transfection: Introduction of DNA into
eukaryotic cells using a vector
Q22.
Explain the principle of recombinant DNA technology.
Ans:
- Combines DNA from two sources
to form recombinant DNA
- Introduced into host for
replication and/or expression
- Tools: restriction enzymes,
ligase, vectors, host cells
Q23.
Name three applications of recombinant DNA technology in medicine.
Ans:
1.
Production
of insulin
2.
Production
of growth hormones and interferon
3.
Gene
therapy for inherited disorders
Q24.
How is human growth hormone produced using biotechnology?
Ans:
- hGH gene inserted into E.
coli plasmid vector
- Bacteria express the protein
- Protein purified using
chromatography for medical use
Q25.
What are the advantages of recombinant proteins over animal-derived proteins?
Ans:
- Pure and safer
- Large-scale production
- Less risk of infection or
immunogenicity
Q26.
Explain the role of multiple cloning sites (MCS).
Ans:
- Region containing several
unique restriction enzyme sites
- Allows flexible insertion of
DNA fragments in a vector
Q27.
What is a palindromic sequence? Give an example.
Ans:
- Reads the same 5’→3’ on both
strands
- Example: GAATTC / CTTAAG
(recognized by EcoRI)
Q28.
How are recombinant colonies identified using antibiotic selection?
Ans:
- Only transformed cells survive
in medium with specific antibiotic
- Non-transformed cells die
- Selective marker ensures identification
of recombinants
Q29.
Explain the importance of Taq polymerase in PCR.
Ans:
- Heat-stable enzyme from Thermus
aquaticus
- Synthesizes DNA at high
temperatures without denaturing
- Enables repeated PCR cycles
efficiently
Q30.
Describe how DNA is visualized after electrophoresis.
Ans:
- Stain gel with ethidium
bromide
- DNA fluoresces orange under UV
light
- Band size compared with a DNA
ladder for estimation
Q31.
What is a vector? Give two examples.
Ans:
- DNA molecule used to carry
foreign gene into host
- Examples: plasmids (pBR322),
bacteriophage (λ phage)
Q32.
How is recombinant DNA inserted into a host?
Ans:
- Bacterial hosts: transformation
(CaCl₂, heat-shock)
- Eukaryotic hosts: transfection
(liposomes, electroporation, gene gun)
Q33.
Explain how insulin A and B chains are combined.
Ans:
- A and B polypeptide chains
produced separately in E. coli
- Purified and chemically
combined via disulfide bond formation
- Functional insulin is obtained
Q34.
Mention any three tools used in genetic engineering.
Ans:
1.
Restriction
enzymes
2.
DNA
ligase
3.
Cloning
vectors
4.
PCR
and gel electrophoresis
Q35.
How does downstream processing ensure product purity?
Ans:
- Removal of cells and media
components
- Filtration, chromatography, and
crystallization
- Quality control ensures safety
and efficacy
Q36.
Explain why sterile conditions are necessary in a bioreactor.
Ans:
- Prevents contamination by
unwanted microbes
- Ensures maximum yield and
purity of recombinant product
Q37.
Define “recombinant DNA”.
Ans:
- DNA formed by joining DNA from
two different sources
- Can replicate and express in a
host cell
Q38.
What is the importance of cloning in biotechnology?
Ans:
- Amplifies desired gene
- Produces large amounts of
protein product
- Facilitates research, medicine,
and agriculture
Q39.
Explain the difference between expression and cloning vectors.
Ans:
|
Feature |
Cloning Vector |
Expression Vector |
|
Purpose |
Gene replication |
Gene expression as protein |
|
Elements |
Ori, selectable marker |
Ori, marker, promoter |
|
Host |
Bacteria |
Bacteria/Yeast |
Q40.
What are the major applications of PCR?
Ans:
1.
Gene
cloning
2.
DNA
fingerprinting
3.
Pathogen
detection
4.
Sequencing
and diagnostics
Q41.
How is a competent cell prepared in E. coli?
Ans:
- Treat with CaCl₂ at cold
temperature
- Apply brief heat-shock
- Membrane becomes permeable to
plasmid DNA
Q42.
Explain how restriction enzymes are named.
Ans:
- First letter: Genus of bacteria
- Next two letters: Species
- Roman numeral: Order of
discovery
Example: EcoRI → E. coli RY13, 1st enzyme discovered
Q43.
Give two examples of industrial products obtained using biotechnology.
Ans:
1.
Ethanol
(fermentation)
2.
Antibiotics
(penicillin, streptomycin)
Q44.
Explain the difference between blunt-end ligation and sticky-end ligation.
Ans:
- Sticky ends: Complementary overhangs,
easier ligation
- Blunt ends: No overhangs, less efficient
ligation
Q45.
Describe how DNA fragments are amplified using PCR.
Ans:
- DNA denatured at 95°C
- Primers anneal at 55°C
- Taq polymerase extends strands
at 72°C
- Repeated cycles produce
millions of copies
Q46.
How are recombinant colonies screened using blue-white screening?
Ans:
- Plasmid has lacZ gene
- Insertion of foreign DNA
inactivates lacZ
- White colonies = recombinant,
Blue = non-recombinant (X-gal assay)
Q47.
Explain the role of ethidium bromide in gel electrophoresis.
Ans:
- Intercalates between DNA bases
- Fluoresces under UV light
- Visualizes DNA bands after
electrophoresis
Q48.
What is the difference between prokaryotic and eukaryotic expression systems?
Ans:
|
Feature |
Prokaryotic |
Eukaryotic |
|
Example |
E. coli |
Yeast, Mammalian cells |
|
Post-translational modification |
None |
Yes (glycosylation) |
|
Protein folding |
Simple |
Complex |
Q49.
Mention the three important properties of a cloning vector.
Ans:
1.
Origin
of replication (Ori)
2.
Selectable
marker
3.
Unique
restriction sites (cloning sites)
Q50.
Describe the steps of downstream processing.
Ans:
1.
Cell
removal:
Filtration or centrifugation
2.
Purification: Chromatography, precipitation
3.
Formulation: Concentration, stabilization
4.
Quality
control: Test for
purity and activity
