Applications of Recombinant DNA Technology

APPLICATIONS
OF
BIO-TECHNOLOGY

HUMAN INSULIN
Human insulin has two polypeptide chains
as A with 21 and B with 30 amino acids,
they are connected through disulphide
bonds.
It occur as proinsulin having a chain C of
33 amino acids, which is converted into
insulin on maturity by dissociation of
chain C.
Banting and Best in 1921, pure insulin
from dog pancreas extract. It was
different than human insulin and its ….

injection to man caused many undesirable
side effects.
In human, DNA sequence codding for
chain A and B is found at top of short arm
of 11th chromosome.
It is of 153 nucleotides, 63 of chain A and
90 of chain B.
It is isolated and inserted into vector
(plasmid pBR322) of E. coli bacteria and
under rDNA technology chains are formed
and joined later to form pure and safe
human insulin.

VACCINE PRODUCTION
 Vaccine is introduction of a mild form of an
antigen into a body to enhance antibody
production and boost immunity to encounter
future infection of that antigen.
 It is done by removing the virulent gene from
desired antigen keeping its ability of
stimulating antibody production by genetic
engineering.
 It is also done by transferring antigen gene
using plasmid vectors to eukaryotes to
produce transgenic plant as potato.
 It can be consumed to develop immunity.

GENE THERAPY
In this process, genes for any
congenital disease are identified and
either they are replaced by a normal
gene, or their expression is suppressed
by increased expression of another
introduced harmless genes by use of
genetic engineering.
e.g. – Some people are affected by
SCID due to defective gene producing
ADA leading to non-functional T-cells.

SCID = Severe Combined Immuno
Deficiency.
ADA = Adenodine Deaminase
Solution for SCID :-
The patient now need normal ADA.
It can be cured by bone marrow
transplantation.
It can also be cured by gene therapy by
replacing the defective ADA making
gene with normal ADA making gene as
follows :-

Identification of normal ADA gene
Its isolation by genetic engineering
Introduction into plasmid vector
It is inserted into E. coli with DNA of
genetically disabled retrovirus
Cloning is allowed, cloned ADA gene is
incorporated into retrovirus

Retrovirus infect blood cells and
transfer ADA gene to them
These cells are cultured, presence of
active ADA gene is ensured
These cells are introduced into affected
child (SCID patient)
T-cells are isolated from her or her
immune system became active

Genetically Modified Crops – Bt Crops
 Bt. Crops are genetically modified crops
containing a varieties of toxins from a bacterium
Bacillus thuringiensis with herbicide tolerance.
 It is done by introducing individual toxin genes
cloned from bacteria through genetic
engineering into desired plants as Bt. Cotton.
 Advantages of Bt – crops
 They are pest resistant, hervicide tolerant.
 They can tolerate cold, drought and salinity.
 They provide more nutritious food.
 They reduce soil and ground water pollution.
 They reduce post harvest losses.

TRANSGENIC ANIMALS
These are genetically modified animals
having one or more foreign gene(s).
Reasons for Producing TA
To identify normal behaviours of genes
and roles of substances they produce.
To know cause and treatment of
diseases.
To provide contents of medicines,
vaccines.
 For other important biological studies.

Production of TA
Desired gene is microinjected into
pronucleus of a reproductive cell of
desired animal.
The modified cell is cultured to specific
embryonic stage and transferred into a
recipient female.
It may also be done by stem cells
(totipotent animal cells).
Totipotent cell is a cell with capacity to
produce entire animal.

Advantages of TA
Selective breeding.
Quality and disease resistance.
It is used for nutritional supplements
and medicines.
It has some industrial applications.
It may be used for biological studies.

MOLECULAR DIAGNOSIS
PCR – Polymerased Chain Reaction.
It is used to detect very low
concentration of bacteria and viruses.
It is possible at a very early stage of
the disease when, other pathological
tests generally not yield result.
It can be done from a small amount of
genetic material of pathogen.
Detection of AIDS is done by this,
even in asymptomatic carriers.

DNA probe is a piece of single stranded DNA or
RNA, tagged with radioactive (Probe) or
fluorescent marker
It is allowed to be hybridised to its
complementary DNA in a clone of cells
Hybridisation is detected by autoradiography
Hybridisation not occur with muted gene as it
has no complementary gene with probe

ELISA – Enzyme Linked Immuno Sorbent
Assay.
It is based on the principle of antigen –
antibody interaction.
It detects the infection of pathogen by
detecting the presence of its antigen or
presence of antibodies against it.
RFLA – Restriction Fragment Length
Analysis
It is used for antenatal diagnosis to identify
genetic disorders of foetus.

About 35 diseases can be identified by this
till date.
It can be done much before birth and the
child birth may be avoided.
Monoclonal Antibodies
 These are homogeneous immunological
reagents of defined specificity and may be
used for diagnosis and screening of infective
diseases.
 They are produced by hybridoma technology
by fusing lymphocyte and myeloma
(cancerous) cell.

THERAPUTIC AGENTS
These are proteins with potential as
pharmaceutical agents produced by
genetic engineering.
Some examples are :-
Human growth hormone obtained from
E. coli is used to treat dwarfness.
Interferons produced from E. coli are
used to treat viral diseases and cancer.
Interleukins produced by E. coli are
used to stimulate immune system.

PROTEINS THROUGH MILK
Desired human gene is linked to specific genes of an
animal that are active only in its mammary gland
Egg of the animal is then injected with the linked
gene
The transgenic animal produced has linked genes in
all its body cells
Human protein is then extracted from milk of
transgenic animal

EXAMPLES OF PROTEINS THROUGH MILK
PROTEIN ANIMAL USE
Antithrombin
III
Goat Reduce amount of blood
needed in surgery
Blood clotting
Factor VIII, IX
Goat, Pig,
Sheep
Treat Haemophilia
Lactoferrin Cow Treat Emphysema and
cystic fibrosis
Alpha – I –
antitrypsin
Sheep Natural antibiotic used in
coronary surgery
Lysostaphin Cow An antibacterial agent to
prevent mastitis in cows

BIOREMEDIATION
 This is a process of using living organisms to
remove contaminants, pollutants or unwanted
substances from soil or water.
 It can remove herbicides, pesticides,
refregerants, solvents etc..
 Many enzymes are required for this
degradation or removal.
 Genes for these enzymes occur on plasmids or
chromosomal DNA.
 Genetically engineered strains of bacteria
Pseudomonas spp. have been produced to
synthesize these enzymes.

BIO – SAFETY ISSUES
These are some safety measures to be
taken to overcome many hazards tend to be
caused by genetically modified organisms
before their commercial use.
BIOPATENT
It is govt. granted right to an inventor
preventing others from its commercial use.
BIOPYRACY
It is violation of patents with out concern
of or compensation to its holder.

BIO WAR
 It is the use of biological agents to cause or
tend to cause casualties in man, animals or
plants.
 The biological agent used is a bio-weapon.
 Bio-weapons are of low cost & more harmful
than conventional weapons / chemicals.
 Their detection is very difficult as are
microscopic and needed in less quantities.
 Rats and their plague were used to infect
Tartar soldiers at Port of Kaffa (Ukrain) in
1936.
 Anthrax used in U. S. A. In September 2001.

BIOETHICS
 It is a set of moral principles or values in use
of genetic engineering and living beings.
 It has been done to obey standards because :-
 Life of animal is disrespected in
experiment.
 Introduction of foreign genes changes
individuality of animals.
 Introduction of human genes into animals
affects integrity of being human.
 Risk of biotechnological changes are
immense.

DISADVANTAGES OF BIO-TECH.
Health – toxicity, food quality, allergy,
pathogen and drug resistance.
Environment – persistent modified
genes, affects on others, side effects.
Agriculture – altered nutritional
value.
General – high cost, ethical issues, loss
of familiarity.
Genetic Pollution – Gene transfer
may lead to genetic pollution.

Applications of Recombinant DNA Technology

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