Biotechnology 1634292871772

NEET- 28
BIOTECHNOLOGY; PRINCIPLES,
PROCESSES AND APPLICATIONS
What is Biotechnology?
Biotechnology is the techniques of using live organisms or their

enzymes for products and processes useful to humans.
European Federation of Biotechnology (EFB) defines

Biotechnology as ‘the integration of natural science and
organisms, cells, parts thereof, and molecular
analogues for products and services’.
Microbe-mediated processes (making
curd, bread, wine etc).
In vitro fertilization
(‘test-tube’ baby programme)
Biotechnology
Synthesis and using of a gene
deals with
Preparation of DNA vaccine
Correcting a defective gene

Sexual reproduction vs Asexual reproduction
•Fusion of two
Sexual different gametes
•Creates variation and
Reproduction unique combinations
of genetic makeup
•Involves only
Asexual single parent
Reproduction •Preserves the
genetic identity of
species
11.1 Principles of Biotechnology
Basic steps in genetically modifying an organism
1. Identification of DNA with desirable genes
2. Introduction of the identified DNA into the

host
3. Maintenance of introduced DNA in the host

and transfer of the DNA to its progeny.
1. Identification of DNA with desirable genes

• Traditional hybridisation techniques lead to
inclusion and multiplication of undesirable
genes & desirable genes.
• In genetic engineering, only desirable genes
are introduced.
2. Introduction of the identified

DNA into the host
• A vector DNA such as plasmid is used
to deliver an alien piece of DNA into
the host organism.
3. Maintenance of introduced DNA in the host and

transfer of the DNA to its progeny.
• A piece of alien DNA has no the sequence called Origin of
replication (ori) needed for starting replication. So, it
cannot multiply itself in the progeny cells of the organism.
• Hence alien DNA is integrated into the recipient genome (it
has ori). It multiplies & inherits along with host DNA.
• The process of joining and inserting a foreign

piece of DNA into a host organism to produce
new genetic combinations is called
recombinant DNA technology.
• First recombinant DNA (rDNA) was produced by Stanley Cohen Herbert Boyer
Stanley Cohen & Herbert Boyer (1972).
• They isolated an antibiotic resistance gene
(piece of DNA) from a plasmid of Salmonella
typhimurium. It was linked with a plasmid
vector and transferred into E. coli. As a result,
the gene was expressed & multiplied in E. coli. Salmonella typhimurium
11.2
11.2 TOOLS OF RECOMBINANT DNA TECHNOLOGY
Restriction
Enzymes Cloning Competent
(‘molecular Vectors Host
scissors’)
11.2.1. Restriction Enzymes (‘molecular scissors’)
• These are the enzymes which cut DNA

at specific sites into fragments.
• They belong to a class of enzymes
called nucleases.
• In 1963, two enzymes responsible for

restricting growth of bacteriophage in E.
coli were isolated. One enzyme added
methyl groups to DNA. The other
(restriction endonuclease) cut DNA.
• More than 900 restriction enzymes have
been isolated from over 230 strains of
bacteria.
11.2.1. Restriction Enzymes
• First letter indicates genus.

• Second two letters indicate
species of the prokaryotic cell
from which they were
isolated.
• E.g. EcoRI comes from E. coli
RY 13.
Exonucleases
• They remove nucleotides from
the ends of the DNA.
Endonucleases
• They cut at specific positions
within DNA.
• They bind to specific recognition
sequence of the DNA and cut the
two strands at specific points.
• First restriction endonuclease is

Hind II.
• It cuts DNA by recognizing a specific
sequence of six base pairs. This is
called the recognition sequence for
Hind II.
Recognition
site of Hind II Recognition sites of various types of
restriction endonucleases
• Restriction endonuclease recognizes a

specific palindromic nucleotide
sequences. It is a sequence of base
pairs that read the same on two
strands in 5' → 3‘ and in 3' → 5'
directions.
5' —— GAATTC —— 3'

3' —— CTTAAG —— 5' Recognition sites of various types of
restriction endonucleases
• Restriction enzymes cut the

strand a little away from the
centre of palindrome sites,
but between the same two
bases on the opposite
strands. This leaves single
stranded overhanging
stretches at the ends. They
are called sticky ends.
• Sticky ends form H-bonds with

their complementary cut
counterparts. This stickiness
facilitates action of DNA ligase.
• When cut by the same
restriction enzyme, the DNA
fragments have the same kind
of sticky-ends. They are joined
by DNA ligases.
• Sticky ends form H-bonds with

their complementary cut
counterparts. This stickiness
facilitates action of DNA ligase.
• When cut by the same
restriction enzyme, the DNA
fragments have the same kind
of sticky-ends. They are joined
by DNA ligases.
11.2.2. Cloning Vectors
• It is a DNA molecule that can carry a foreign DNA segment and replicate
inside the host cells.
• E.g. Plasmids, bacteriophages, etc.
11.2.2. Cloning Vectors
• Plasmids are autonomously replicating circular extra-chromosomal DNA of
bacteria. Some plasmids have only 1-2 copies per cell. Others have 15-100
copies per cell.
• Bacteriophages (high number per cell) have very high copy numbers of their
genome within the bacterial cells.
TOOLS OF RECOMBINANT DNA TECHNOLOGY
2. Cloning Vectors
• When the cloning vectors are multiplied in the host, the linked piece of DNA
is also multiplied to the numbers equal to the copy number of the vectors.
www.bankofbiology.com
2. Cloning
Features required for cloning into a vector
Vectors
• Restriction sites: Hind

III, EcoR I, BamH I,
A. Origin of replication (ori) Sal I, Pvu II, Pst I, Cla
B. Selectable marker I.
(Marker gene) • Ori
• Antibiotic resistance
C. Cloning sites genes: ampR & tetR.
D. Vectors for cloning genes • Rop: codes for the
proteins involved in
in plants & animals the replication of
plasmid.
2. Cloning
Vectors
A. Origin of replication (ori)

• It is a sequence where replication starts.
• A piece of DNA linked to ori can replicate within
the host cells.
• It also controls the copy number of linked DNA. So,
for getting many copies of target DNA, it should be
cloned in a vector whose origin support high copy
number.
2. Cloning
Vectors
B. Selectable marker (marker gene) E. Coli

cloning
• It is a gene that helps to select transformants and vector
eliminate the non-transformants. pBR322
• If a piece of DNA is introduced in a host bacterium,

it is called transformation. Such bacterium is
transformant.
• If transformation does not take place, it is non-
transformant.
2. Cloning
Vectors
B. Selectable marker (marker gene) E. Coli

cloning
vector
pBR322
• Selectable markers of E. coli include genes
encoding resistance to antibiotics like ampicillin,
chloramphenicol, tetracycline, kanamycin, etc.
• Normal E. coli cells have no resistance against
these antibiotics.
2. Cloning
Vectors
C. Cloning sites
• To link the alien DNA, the vector needs a

single or very few recognition sites for
restriction enzymes.
• More than one recognition sites generates
several fragments. It complicates the gene
cloning.
E. Coli cloning vector pBR322
2. Cloning
Vectors
C. Cloning sites
• Ligation of alien DNA is carried out at a
restriction site present in one of the two
antibiotic resistance genes.
• E.g. ligation of a foreign DNA at Bam H I site of
tetracycline resistance gene in vector pBR322.
As a result, recombinant plasmid is formed.
• If ligation does not occur, it is called non-
recombinant plasmid. E. Coli cloning vector pBR322
2. Cloning
Vectors
C. Cloning sites
• When a foreign DNA is inserted within a gene of
bacteria, that gene is inactivated. It is called
insertional inactivation. Here, the recombinant
plasmids lose tetracycline resistance due to
insertion of foreign DNA.
• When the plasmids are introduced into E. coli
cells, 3 types of cells are obtained:
2. Cloning
Vectors
C. Cloning sites
1. Non-transformants: They have no plasmid. So
they are not resistant to either tetracycline or
ampicillin.
2. Transformants with non-recombinant plasmid:
They are resistant to both tetracycline &
ampicillin.
3. Transformants with recombinant plasmid: They
are resistant only to ampicillin.
2. Cloning
Vectors
C. Cloning sites
• Recombinant plasmids can be selected out from

non-recombinant ones by plating the transformants
on ampicillin medium. Then the transformants are
transferred on tetracycline medium.
• The recombinants grow in ampicillin medium but
not on tetracycline medium. But, non-recombinants
grow on medium containing both the antibiotics.
2. Cloning
Vectors
C. Cloning sites
• Thus, one antibiotic resistance gene helps to

select the transformants.
• The inactivated antibiotic resistance gene
helps to select recombinants.
2. Cloning
Vectors
C. Cloning sites
• But this type of selection of recombinants is a

difficult procedure because it needs simultaneous
plating on 2 plates having different antibiotics.
• So, alternative selectable markers have
developed based on their ability to produce
colour in presence of a chromogenic substrate.
2. Cloning
Vectors
C. Cloning sites
• In this, a recombinant DNA is inserted into the
coding sequence (gene) of an enzyme, b-
galactosidase. So the gene is inactivated (insertional
inactivation). Such colonies do not produce colour.
These are identified as recombinant colonies.
• If the plasmid in bacteria have no an insert, it gives
blue coloured colonies in presence of chromogenic
substrate.
2. Cloning
Vectors
C. Cloning sites
• In this, a recombinant DNA is inserted into the
coding sequence (gene) of an enzyme, b-
galactosidase. So the gene is inactivated (insertional
inactivation). Such colonies do not produce colour.
These are identified as recombinant colonies.
• If the plasmid in bacteria have no an insert, it gives
blue coloured colonies in presence of chromogenic
substrate.
2. Cloning
Vectors
D. Vectors for cloning genes in plants & animals
• Genetic tools of some pathogens is

transformed into useful vectors for Agrobacterium tumefaciens - SEM
delivering genes to plants & animals. E.g.
 Agrobacterium tumefaciens (a pathogen
of many dicot plants). A
retrovirus
 Retroviruses
2. Cloning
Vectors

 Agrobacterium tumefaciens can deliver a piece of
DNA (T-DNA) to transform normal plant cells into a
tumor. These tumor cells produce the chemicals
required by the pathogen.
 Tumor inducing (Ti) plasmid of A. tumefaciens is
modified into a cloning vector which is not
pathogenic but can use the mechanisms to deliver
genes of interest into plants.
2. Cloning
Vectors

 Agrobacterium tumefaciens can deliver a piece of
DNA (T-DNA) to transform normal plant cells into a
tumor. These tumor cells produce the chemicals
required by the pathogen.
 Tumor inducing (Ti) plasmid of A. tumefaciens is
modified into a cloning vector which is not
pathogenic but can use the mechanisms to deliver
genes of interest into plants.
2. Cloning
Vectors
 Retroviruses in animals can transform

normal cells into cancerous cells.
 So they are used to deliver desirable genes
into animal cells.
3. Competent Host (For Transformation with Recombinant DNA)
• Since DNA is a hydrophilic molecule, it

cannot pass through cell membranes.
• So the bacterial cells must be made
‘competent’ to take up alien DNA or
plasmid.
• For this, bacterial cells are treated with a
specific concentration of a divalent cation
(e.g. calcium). So DNA enters the
bacterium through pores in cell wall.
• Such cells are incubated with

recombinant DNA on ice.
• Then they are placed briefly at 420C (heat
shock) and put them back on ice.
• This enables the bacteria to take up the
recombinant DNA.
Other methods to introduce alien DNA into host cells
• Micro-injection: Recombinant DNA is directly injected into nucleus of an animal cell.

• Biolistics (gene gun): Cells are bombarded with high velocity micro-particles of gold or
tungsten coated with DNA. This method is suitable for plants.
• ‘Disarmed pathogen’ vectors: When it infects the cell, transfer recombinant DNA into
the host.
PROCESSES OF RECOMBINANT DNA TECHNOLOGY
1. Isolation of the Genetic Material (DNA)
2. Cutting of DNA at Specific Locations
3. Amplification of Gene of Interest using PCR
4. Insertion of Recombinant DNA into Host Cell
5. Obtaining the Foreign Gene Product
6. Downstream Processing
• Treat bacterial cells/plant or animal

tissue with enzymes like lysozyme
(bacteria), cellulase (plants), chitinase
(fungus) etc.
• The cell is broken releasing DNA & other
macromolecules (RNA, proteins,
polysaccharides & lipids).
• RNA is removed by treating with ribonuclease. Proteins are removed by treating
with protease. Other molecules are removed by appropriate treatments.
• When chilled ethanol is added, purified DNA precipitates out as fine threads in
the suspension.
• Purified DNA is incubated with the restriction enzyme. As a result, DNA digests.
• DNA fragments are separated by a technique called gel electrophoresis.
• Agarose gel electrophoresis is employed to check the progression of a restriction
enzyme digestion.
• DNA is negatively charged. So it moves towards the anode.
• DNA fragments are separated according to their size through sieving effect of agarose
gel (polymer from sea weeds). Smaller sized fragment move farther.
• The process is repeated with the vector DNA also.
• DNA fragments can be seen as bright orange coloured bands when they are
stained with ethidium bromide and exposed to UV radiation.
• DNA bands are cut out from agarose gel. This is called elution.
• The cut-out gene of interest from source DNA and cut vector are mixed and ligase
is added. It creates recombinant DNA.
• Polymerase Chain Reaction (PCR) is the

synthesis of multiple copies of the gene
of interest in vitro using 2 sets of primers
& the enzyme DNA polymerase.
• Primers are small chemically synthesized
oligonucleotides that are complementary
to the regions of DNA.
1. Denaturation
STEPS OF
2. Annealing
PCR
3. Extension
Steps of PCR 1. Denaturation
• It is the heating of target DNA (gene of

interest) at high temperature (940 C) to
separate the strands.
• Each strands act as template for DNA
synthesis.
Steps of PCR 2. Annealing
• It is the joining of the two primers (at

520 C) at the 3’ end of the DNA
templates.
Steps of PCR 3. Extension
• It is the addition of nucleotides to the primer

using a thermostable DNA polymerase called
Taq polymerase. It is isolated from a
bacterium, Thermus aquaticus. It remains
active in high temperature during the
denaturation of double stranded DNA.
• Through continuous replication, the DNA
segment is amplified up to 1 billion copies.
• The amplified fragment can be used to ligate
with a vector for further cloning.
PCR AT A
GLANCE
4. Insertion of Recombinant DNA into Host Cell /organism
• Using any methods, the ligated DNA is introduced into recipient (host) cells. They
take up DNA from its surrounding.
• If a recombinant DNA bearing ampicillin resistant gene is transferred into E. coli cells,
the host cells become ampicillin-resistant cells.
• If the transformed cells are spread on agar plates containing ampicillin, only
transformants will grow. Untransformed recipient cells will die.
• The ultimate aim of recombinant DNA technology is to produce a desirable

protein.
• If a protein encoding foreign gene is expressed in a heterologous host, it is called
a recombinant protein.
• Cells with foreign genes can be grown in laboratory. The cultures are used to extract
desired protein and purify it by using separation techniques.
• The cells can also be multiplied in a continuous culture system. Here, the used
medium is drained out from one side while fresh medium is added from the other. It
maintains the cells more physiologically active and so produces a larger biomass. It
yields more desired protein.
Bioreactors
• These are the vessels in which raw materials
are biologically converted into specific
products, enzymes etc. using microbial, plant,
animal or human cells.
• Bioreactors are used to produce large
quantities of products. They can process 100-
1000 litres of culture.
• A bioreactor provides optimal growth
conditions (temperature, pH, substrate, salts,
vitamins, oxygen) to get desired product.
Bioreactors
• Most commonly used bioreactors
are of stirring type (stirred-tank
reactor).
• It is usually cylindrical or with a
curved base to facilitate the
mixing of the reactor contents.
The stirrer facilitates even mixing
and oxygen availability.
Alternatively air can be bubbled
through the reactor.
Bioreactors
Parts of a Bioreactor
• An agitator system
• An oxygen delivery system
• A foam control system
• A temperature control system
• pH control system
• Sampling ports (for periodic
withdrawal of the culture)
6. Downstream Processing
• It is a series of processes
such as separation and
purification of products after
the biosynthetic stage.
• The product is formulated
with suitable preservatives.
Such formulation undergoes
clinical trials and strict
quality control testing.
Biotechnology: Applications
1. Biopharmaceuticals
2. Therapeutics
3. Diagnostics
4. Genetically modified crops
5. Processed food
6. Bioremediation
7. Waste treatment
8. Energy production
Biotechnology: Applications
Provide best catalyst in the form of
improved organism usually a
microbe or pure enzyme.
3 critical research
Create optimal conditions through
areas of engineering for a catalyst to act.
Biotechnology
Downstream processing
technologies to purify
protein/organic compound.
APPLICATIONS IN AGRICULTURE
3 options for increasing food production
Agro-chemical based
agriculture
Organic agriculture
Genetically Modified Organisms (GMO)
are the plants, bacteria, fungi & animals
Genetically engineered
whose genes are altered by
crop-based agriculture
manipulation.
Advantages of genetic modification in plants
It makes crops more tolerant to abiotic stresses
(cold, drought, salt, heat etc).
Pest-resistant crops reduce use of chemical
pesticides.
It helps to reduce post harvest losses.
It increases efficiency of mineral usage by plants
(it prevents early exhaustion of soil fertility).
It enhances nutritional value of food. E.g. Golden
rice (Vitamin ‘A’ enriched rice).
GM is used to create tailor-made plants to supply
alternative resources (starches, fuels,
pharmaceuticals etc.) to industries.
Pest Resistant Plants
• Pest Resistant Plants act as

bio-pesticide.
• It reduces the need for
insecticides.
• E.g. Bt cotton, Bt corn, rice,
tomato, potato, soyabean etc.
Bt Cotton
• Some strains of Bacillus thuringiensis have

proteins that kill insects like coleopterans
(beetles) lepidopterans (tobacco budworm,
armyworm) & dipterans (flies, mosquitoes). Bacillus thuringiensis
• B. thuringiensis forms a toxic insecticidal
protein (Bt toxin) crystal during a particular
phase of their growth. It does not kill the
Bacillus as it exists as inactive protoxins.
Bt Cotton
• When an insect ingest the inactive

toxin, it is converted into active
toxin due to the alkaline pH of the
gut which solubilise the crystals.
• The toxin binds to the surface of
midgut epithelial cells and creates
pores. It causes cell swelling and
lysis and death of the insect.
Bt Cotton
• Bt toxin genes were isolated from B.

thuringiensis and incorporated into crop
plants such as cotton.
• Most Bt toxins are insect-group specific.
• The toxin is coded by a gene cryIAc
named cry.
• E.g. proteins encoded by genes cryIAc &
cryIIAb control the cotton bollworms
that of cryIAb controls corn borer.
Nematode resistance in tobacco plants
• A nematode Meloidogyne incognita

infects the roots of tobacco plants
causing a reduction in yield.
• It can be prevented by RNA
interference (RNAi) strategy.
• RNAi is a method of cellular defense
in all eukaryotes. It prevents
translation of a specific mRNA Meloidogyne incognita
(silencing) due to a complementary
dsRNA molecule.
Nematode resistance in tobacco plants
• The source of complementary RNA is from an infection by RNA

viruses or mobile genetic elements (transposons) that
replicate via an RNA intermediate.
• Using Agrobacterium vectors, nematode-specific genes (DNA)
is introduced into host plant. It produces both sense & anti-
sense RNA in host cells. These RNA’s are complementary. So
they form a double stranded (ds) RNA. It initiates RNAi and
silence the specific mRNA of nematode.
• Thus the parasite cannot survive in a transgenic host
expressing specific interfering RNA. Nematode infected tobacco
root
APPLICATIONS IN MEDICINE
• Recombinant DNA technology helps for the

mass production of safe and more effective
therapeutic drugs.
• The products from non-human sources
induce unwanted immunological responses.
But recombinant therapeutics does not have
such problems.
• At present, about 30 recombinant
therapeutics have been approved. Of these,
12 are being marketed in India.
1. Genetically Engineered Insulin

2. Gene Therapy
3. Molecular Diagnosis
• Insulin is used to manage adult-

onset diabetes.
• Insulin from the pancreas of
animals (cattle & pigs) causes
allergy or other types of reactions
to the foreign protein.
• Now, it is possible to produce
human insulin using bacteria.
• Insulin consists of 2 short polypeptide chains

(chain A & chain B) that are linked by disulphide
bridges.
• In mammals, insulin is synthesized as a pro-
hormone. It needs processing to become mature
and functional hormone.
• The pro-hormone contains an extra stretch
called the C peptide. This is removed during
maturation into insulin.
• In 1983, Eli Lilly, (an American company)

prepared two DNA sequences
corresponding to A & B chains of human
insulin.
• They were introduced in plasmids of E.
coli to produce insulin chains. Chains A &
B were combined by creating disulfide
bonds to form human insulin.
2. Gene Therapy
• It is a method to correct a gene

defect in a child/embryo.
• Here, genes are inserted into a
person’s cells and tissues to treat a
hereditary disease. It compensates
for the non-functional gene.
2. Gene Therapy
• First clinical gene therapy (1990) was given to a 4-

year old girl with adenosine deaminase (ADA)
deficiency.
• It is caused due to deletion of the gene for
adenosine deaminase (the enzyme for immune
system to function).
• Treatment by bone marrow transplantation or
enzyme replacement therapy (injection of ADA) is
not completely curative. Ashanti De Silva: First Clinical
gene therapy patient
2. Gene Therapy
Gene therapy for ADA deficiency

• Collect lymphocytes from the patient’s
blood and grow in a culture →
Introduce a functional ADA cDNA into
lymphocytes (using a retroviral vector)
→ They are returned to the patient.
• This should be periodically repeated as
lymphocytes are not immortal.
Gene therapy for ADA-SCID
If ADA gene from marrow cells is introduced into cells at early embryonic stages, it
could be a permanent cure.
3. Molecular Diagnosis
• Conventional methods (serum

& urine analysis) are not
suitable for early diagnosis of
diseases.
• It is possible by techniques
like Recombinant DNA
technology, PCR & ELISA.
3. Molecular Diagnosis: PCR (Polymerase Chain Reaction)
• Presence of a pathogen is normally suspected

only based on symptoms. By this time, the
concentration of pathogen is already very high
in the body.
PCR Thermal cycler
• However, very low concentration of a bacteria
or virus can be detected by amplification of
their nucleic acid by PCR.
PCR Tubes
3. Molecular Diagnosis: PCR (Polymerase Chain Reaction)
Uses of PCR
• To detect HIV in suspected AIDS patients.
• To detect mutations in genes in cancer patients.
• To identify many other genetic disorders.
• A single stranded DNA or RNA, tagged with a radioactive molecule (probe) is

hybridized to its complementary DNA in a clone of cells. It is followed by detection
using autoradiography.
• The clone having mutated gene will not appear on photographic film, because the
probe will not have complementarity with the mutated gene.
3. Molecular Diagnosis: ELISA (Enzyme Linked Immuno-Sorbent Assay)
• ELISA is based on the principle of

antigen-antibody interaction.
• Infection by pathogen can be
detected by the presence of
antigens (proteins, glycoproteins
etc.) or by detecting the
antibodies synthesized against
the pathogen.
TRANSGENIC ANIMALS
• These are the animals whose genome

Mice Rabbit has been altered by introduction of an
extra (foreign) gene by manipulation.
• E.g. Transgenic rats, rabbits, pigs,
Sheep Pigs sheep, cows and fish.
• Over 95% of the transgenic animals
are mice.
Cows Fish
TRANSGENIC ANIMALS
Benefits of Transgenic animals
1. To study the regulation of genes on normal
physiology & development
2. To Study the contribution of genes in the development of

a disease
3. Biological products
4. Vaccine safety testing
5. Chemical safety testing (toxicity testing)

TRANSGENIC ANIMALS
1. To study regulation of genes on normal physiology & development
• E.g. Study of complex factors such as insulin-like

growth factor.
• Genes (from other species) that alter formation
of this factor are introduced and the biological
effects are studied.
• This gives information about the biological role
of the factor in the body.
Insulin-like growth factor 1
TRANSGENIC ANIMALS
2. To study the contribution of genes in the development of a disease
• Transgenic models help to investigate

new treatments for human diseases.
• E.g. transgenic models for many human
diseases such as cancer, cystic fibrosis,
rheumatoid arthritis and Alzheimer’s.
TRANSGENIC ANIMALS
• Transgenic animals are used to produce

biological products by introducing genes
which codes for a particular product. E.g.
• Human protein (a-1-antitrypsin) used
to treat emphysema.
• Products for treatment of
phenylketonuria (PKU) and cystic
fibrosis etc.
Emphysema
TRANSGENIC ANIMALS
• In 1997, Rosie (first transgenic cow)

produced human protein-enriched milk
(2.4 gm per litre).
• It contains human a-lactalbumin. It is
nutritionally more balanced product for
human babies than natural cow-milk.
TRANSGENIC ANIMALS
4. Vaccine safety testing
• Transgenic mice are used to test

the safety of the polio vaccine.
• If it is reliable, they can replace
the use of monkeys to test the
safety of the vaccine.
TRANSGENIC ANIMALS
5. Chemical safety testing (Toxicity testing)
• Some transgenic animals carry genes which

make them more sensitive to toxic
substances than non-transgenic animals.
• They are exposed to the toxic substances and
the effects studied. It gives immediate
results.
ETHICAL ISSUES
Problems of unpredicted results
• Genetic modification may cause

unpredictable results when such organisms
are introduced into ecosystem.
• Indian Government has set up organizations
like GEAC (Genetic Engineering Approval
Committee), which make decisions about
the validity of GM research and the safety of
GM-organisms for public services.
ETHICAL ISSUES
Biopiracy
• It is the use of bio-resources by multinational

companies and other organizations without
proper authorization from the countries and
people concerned.
• Certain companies got patents for products and
technologies that make use of the genetic
materials, plants etc. that have been identified,
developed and used by farmers and indigenous
people of a country. E.g. Basmati rice, herbal
medicines (turmeric, neem etc.).
ETHICAL ISSUES
Biopiracy
• Basmati rice has unique aroma & flavour.

• India has 27 varieties of Basmati.
• In 1997, an American company got patent
rights on Basmati rice through the US Patent
and Trademark Office. This allowed the
company to sell a ‘new’ variety of Basmati.
This was actually derived from Indian farmer’s
varieties. Indian Basmati was crossed with
semi-dwarf varieties and claimed as a novelty.
Other people selling Basmati rice could be
restricted by patent.
ETHICAL ISSUES
Biopiracy
• Most of the industrialized nations are poor in

biodiversity and traditional knowledge. The
developing and the underdeveloped world
have rich biodiversity and traditional
knowledge related to bio-resources.
ETHICAL ISSUES
Biopiracy
• It has to develop laws to prevent

unauthorized exploitation of bio-
resources & traditional knowledge.
• Indian Parliament has cleared the
second amendment of the Indian
Patents Bill that takes such issues
into consideration, including patent
terms emergency provisions and
research and development initiative.

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NEET- 28

Biotechnology is the techniques of using live organisms or their

European Federation of Biotechnology (EFB) defines

Preparation of DNA vaccine

Correcting a defective gene

1. Identification of DNA with desirable genes

2. Introduction of the identified DNA into the

3. Maintenance of introduced DNA in the host

1. Identification of DNA with desirable genes

2. Introduction of the identified

3. Maintenance of introduced DNA in the host and

• The process of joining and inserting a foreign

• These are the enzymes which cut DNA

• In 1963, two enzymes responsible for

• First letter indicates genus.

• First restriction endonuclease is

• Restriction endonuclease recognizes a

5' —— GAATTC —— 3'

• Restriction enzymes cut the

• Sticky ends form H-bonds with

• Sticky ends form H-bonds with

• Restriction sites: Hind

A. Origin of replication (ori)

B. Selectable marker (marker gene) E. Coli

• If a piece of DNA is introduced in a host bacterium,

B. Selectable marker (marker gene) E. Coli

• To link the alien DNA, the vector needs a

• Recombinant plasmids can be selected out from

• Thus, one antibiotic resistance gene helps to

• But this type of selection of recombinants is a

D. Vectors for cloning genes in plants & animals

• Genetic tools of some pathogens is

D. Vectors for cloning genes in plants & animals

D. Vectors for cloning genes in plants & animals

D. Vectors for cloning genes in plants & animals

 Retroviruses in animals can transform

• Since DNA is a hydrophilic molecule, it

• Such cells are incubated with

• Micro-injection: Recombinant DNA is directly injected into nucleus of an animal cell.

1. Isolation of the Genetic Material (DNA)

2. Cutting of DNA at Specific Locations

3. Amplification of Gene of Interest using PCR

4. Insertion of Recombinant DNA into Host Cell

5. Obtaining the Foreign Gene Product

• Treat bacterial cells/plant or animal

• Polymerase Chain Reaction (PCR) is the

• It is the heating of target DNA (gene of

• It is the joining of the two primers (at

• It is the addition of nucleotides to the primer

• The ultimate aim of recombinant DNA technology is to produce a desirable

• Pest Resistant Plants act as

• Some strains of Bacillus thuringiensis have

• When an insect ingest the inactive

• Bt toxin genes were isolated from B.

• A nematode Meloidogyne incognita

• The source of complementary RNA is from an infection by RNA

• Recombinant DNA technology helps for the

1. Genetically Engineered Insulin

• Insulin is used to manage adult-