🌸Chapter 9
Biotechnology: Principles and Processes
(3Marks)
Q1.
Explain the basic principles of biotechnology.
Ans: The three basic principles are:
1.
Genetic
engineering
– to alter the genetic material (DNA/RNA) for useful products.
2.
Maintenance
of sterile conditions
– to grow only the desired organism.
3.
Downstream
processing – for
purification and formulation of the product.
Q2.
Mention the main steps involved in genetic engineering.
Ans:
1.
Isolation
of genetic material (DNA)
2.
Cutting
of DNA using
restriction enzymes
3.
Ligation
of DNA fragment
into a vector
4.
Introduction
into host cell
(transformation)
5.
Selection
and screening
of recombinants
6.
Large-scale
production and downstream
processing
Q3.
What are restriction enzymes? Give one example and its function.
Ans:
Restriction enzymes are molecular scissors that cut DNA at specific sequences
called restriction sites.
Example: EcoRI recognizes the sequence GAATTC and cuts between G
and A, producing sticky ends.
Q4.
Differentiate between exonuclease, endonuclease, and restriction endonuclease.
Ans:
- Exonuclease: Removes nucleotides from ends
of DNA.
- Endonuclease: Cuts DNA at specific internal
sites.
- Restriction endonuclease: Special type of endonuclease
that cuts DNA at specific palindromic sites.
Q5.
What are sticky ends? Why are they useful in recombinant DNA technology?
Ans: Sticky ends are short single-stranded overhangs created by
restriction enzymes.
They help in joining foreign and vector DNA through complementary base
pairing and ligation.
Q6.
Write short notes on plasmids.
Ans:
Plasmids are circular, double-stranded, self-replicating DNA molecules
in bacteria.
They often carry antibiotic resistance genes and are used as vectors
in genetic engineering.
Q7.
What are the essential features of a cloning vector?
Ans:
1.
Origin
of replication (Ori)
– for self-replication
2.
Selectable
marker – for
selection of recombinants
3.
Cloning
sites – for
insertion of foreign DNA
4.
Small
size – for easy transfer into host cell
Q8.
What is the role of ori (origin of replication) in a cloning vector?
Ans: Ori is the DNA sequence where replication starts. It allows the
inserted DNA to replicate inside the host cell, producing multiple copies.
Q9.
What are selectable markers? Give two examples.
Ans: Selectable markers help identify transformants.
Examples: amp^r (ampicillin resistance) and tet^r (tetracycline
resistance) genes in plasmid pBR322.
Q10.
What is pBR322?
Ans: pBR322 is a plasmid vector developed by Bolivar and
Rodriguez. It contains:
- Ori (origin of replication)
- Selectable markers (amp^r and
tet^r)
- Unique restriction sites for
cloning.
Q11.
What are cloning sites?
Ans: Cloning sites are unique restriction enzyme recognition sequences
on a vector where foreign DNA can be inserted for cloning.
Q12.
Define the term “transformation.”
Ans: Transformation is the process by which a bacterial cell takes up
foreign DNA from its surroundings, making it genetically modified.
Q13.
Explain how recombinant DNA is introduced into the host cell.
Ans: Methods:
1.
CaCl₂
treatment in
bacteria – makes cell membrane permeable.
2.
Microinjection – DNA injected into animal cell
nucleus.
3.
Gene
gun/biolistics
– DNA-coated particles shot into plant cells.
Q14.
What are competent cells? How are they made?
Ans: Competent cells are bacterial cells that can take up DNA.
They are made by treating bacteria with CaCl₂, which makes the cell wall
permeable.
Q15.
What are the three steps of PCR?
Ans:
1.
Denaturation
(95°C): DNA
strands separate.
2.
Annealing
(55°C): Primers
bind to template.
3.
Extension
(72°C): Taq
polymerase synthesizes new DNA strands.
Q16.
Explain the principle of PCR.
Ans: PCR uses Taq polymerase to amplify a specific DNA sequence
through repeated cycles of denaturation, annealing, and extension.
Q17.
What is the role of Taq polymerase in PCR?
Ans: Taq polymerase is a heat-stable DNA polymerase from Thermus
aquaticus that synthesizes DNA at high temperatures during PCR.
Q18.
What is gel electrophoresis?
Ans: It is a technique to separate DNA fragments based on their
size using agarose gel and electric current; smaller fragments
move faster toward the anode.
Q19.
How are DNA fragments visualized after electrophoresis?
Ans: DNA fragments are stained with ethidium bromide and observed
under UV light, where they appear as orange fluorescent bands.
Q20.
What is recombinant DNA technology? Mention its basic steps.
Ans:
It is a process of joining DNA from different sources.
Steps:
1.
Isolation
of DNA
2.
Cutting
by restriction enzymes
3.
Ligation
into vector
4.
Insertion
into host
5.
Selection
and culturing
Q21.
Define palindromic sequence with an example.
Ans:
A palindromic DNA sequence reads the same in 5'→3' direction on both strands.
Example: GAATTC / CTTAAG (recognized by EcoRI).
Q22.
Why are palindromic sequences important in biotechnology?
Ans: Restriction enzymes recognize and cut at palindromic sequences,
enabling precise DNA fragment isolation for cloning.
Q23.
What is a bioreactor?
Ans: A bioreactor is a large vessel that provides controlled
conditions (temperature, pH, oxygen, nutrients) for large-scale microbial or
cell culture to produce recombinant products.
Q24.
Name two types of bioreactors and differentiate them.
Ans:
1.
Stirred-tank
bioreactor:
Uses mechanical agitation.
2.
Airlift
bioreactor:
Uses air bubbles for mixing.
Stirred-tank has impellers; airlift is simpler and avoids mechanical shear.
Q25.
What is downstream processing?
Ans: It includes all steps after fermentation — separation,
purification, quality control, and formulation of the final recombinant product
(e.g., insulin).
Q26.
What is the role of the sparger and agitator in a bioreactor?
Ans:
- Sparger: Introduces sterile air
bubbles.
- Agitator: Ensures uniform mixing and
oxygen distribution.
Q27.
How is recombinant insulin produced?
Ans:
1.
Human
insulin gene inserted into E. coli plasmid.
2.
E.
coli expresses A and B polypeptide
chains.
3.
Chains
purified and combined to form functional insulin.
Q28.
What are the advantages of using recombinant DNA technology in medicine?
Ans:
1.
Production
of safer, pure medicines (e.g., insulin, interferon).
2.
Faster,
large-scale production.
3.
No
risk of allergies from animal proteins.
Q29.
Differentiate between cloning vector and expression vector.
Ans:
- Cloning vector: For gene insertion and
replication.
- Expression vector: For gene insertion and
expression into protein.
Q30.
What is the difference between exons and introns?
Ans:
- Exons: Coding sequences that express
proteins.
- Introns: Non-coding sequences removed
during RNA splicing.
Q31.
Why is E. coli preferred as a host in biotechnology experiments?
Ans:
1.
Fast
growth rate.
2.
Easy
to culture and manipulate.
3.
Genome
well-known.
4.
Readily
takes up recombinant plasmids.
Q32.
What are multiple cloning sites?
Ans:
They are regions in vectors containing several unique restriction enzyme sites,
allowing flexibility for inserting different DNA fragments.
Q33.
What are the major tools of recombinant DNA technology?
Ans:
1.
Restriction
enzymes
2.
Cloning
vectors
3.
Host
organisms
4.
DNA
ligase
5.
PCR
and electrophoresis
Q34.
Describe the process of DNA isolation.
Ans:
1.
Cell
lysis using enzymes.
2.
Removal
of proteins and RNA.
3.
DNA
precipitation with chilled ethanol.
The purified DNA is then used for further manipulation.
Q35.
What are the applications of PCR?
Ans:
1.
Gene
cloning and diagnosis of diseases.
2.
DNA
fingerprinting.
3.
Detection
of pathogens.
4.
Amplification
for sequencing.
Q36.
Why is it necessary to maintain sterile conditions in biotechnological
experiments?
Ans:
To prevent contamination by other microorganisms that can spoil the culture and
reduce yield of the desired product.
Q37.
Explain the principle behind recombinant DNA technology.
Ans:
The principle is combining DNA from two different sources to form
recombinant DNA, which is then inserted into a host for expression.
Q38.
How can we identify recombinant and non-recombinant colonies?
Ans:
Using selectable markers or insertional inactivation, where insertion of
foreign DNA disrupts a marker gene, changing colony color or antibiotic
resistance.
Q39.
What is insertional inactivation?
Ans:
It is a process where insertion of a foreign gene inactivates a marker gene
(like lacZ), helping identify recombinant colonies (white vs blue in
X-gal medium).
Q40.
How are large quantities of recombinant proteins produced?
Ans:
Recombinant host cells are cultured in bioreactors under controlled
conditions to express and multiply the desired protein.
Q41.
Why are heat-shock and CaCl₂ treatment given to bacterial cells?
Ans:
They temporarily make the bacterial membrane permeable, allowing DNA to enter
during transformation.
Q42.
Mention three advantages of recombinant DNA technology.
Ans:
1.
Precise
modification of genes.
2.
Cross-species
gene transfer.
3.
Production
of useful proteins, vaccines, and GM crops.
Q43.
Why is the use of the same restriction enzyme important for cutting vector and
foreign DNA?
Ans:
It ensures that both DNA fragments have complementary sticky ends that
can easily anneal and be joined by ligase.
Q44.
How does a bioreactor help in large-scale production?
Ans:
It maintains optimal temperature, pH, and oxygen levels, allowing
continuous, large-scale culture of recombinant microbes for product synthesis.
Q45.
What are the limitations of traditional hybridization over genetic engineering?
Ans:
Traditional methods are time-consuming, imprecise, and limited to related
species, while genetic engineering is fast and cross-species.
Q46.
Explain how DNA fragments are separated by gel electrophoresis.
Ans:
When electric current is passed, negatively charged DNA moves toward the anode
through agarose gel; smaller fragments move faster, thus separating by size.
Q47.
What is the purpose of downstream processing?
Ans:
To purify, test, and formulate the final recombinant product for safe use in
pharmaceuticals, agriculture, or industry.
Q48.
What is the role of buffers in electrophoresis?
Ans:
Buffers maintain constant pH and provide ions to conduct electric current
during DNA migration in gel electrophoresis.
Q49.
How is recombinant DNA visualized after electrophoresis?
Ans:
By staining with ethidium bromide and observing under UV light, where
DNA bands appear orange.
Q50.
Explain how biotechnology has revolutionized medicine.
Ans:
- Production of recombinant
insulin, interferon, vaccines
- Gene therapy for inherited disorders
- Accurate diagnosis using PCR
→ All ensuring safer and faster treatments.
