RECOMBINANT

DNA TECHNOLOGY

Dr. A.D.NAVEEN KUMAR

CMHS, ADIGRAT UNIVERSITY

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 Overview of DNA Technology
• Definition of terminologies
• Restriction endonucleases, Restriction map,
Chimeric DNA (recombinant DNA)
• Cloning & cloning vectors
• Recombinant libraries
• Genomic library vs cDNA library
• Blotting & visualization techniques
• (Southern blotting, northern blotting, western
blotting)
• DNA nucleotide sequence analysis
• The polymerase chain reaction
• Practical applications of recombinant DNA
technology
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 Introduction
• Biotechnology is an interdisciplinary
science including not only biology and also
subjects like mathematics, physics,
chemistry and engineering.

• It is a blend of various technologies applied

together to living cells for the production of a
particular product or for improving upon it.

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Recombinant DNA technology is one of the
recent advances in biotechnology developed
by two scientists named Boyer and Cohen in
1973.

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 Recombinant DNA and
Gene Cloning
 Recombinant DNA (rDNA) is a form of artificial
DNA that is created by combining two or more
sequences that would not normally occur together
through the process of gene splicing.

 Recombinant DNA technology is a technology

which allows DNA to be produced by artificial
means. The procedure has been used to change
DNA in living organisms and may have even more
practical uses in the future.

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 Recombinant DNA technology works by
taking DNA from two different sources and
combining that DNA into a single molecule.

 Recombinant DNA technology only becomes

useful when that artificially-created DNA is
reproduced. This is known as DNA cloning.

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7
 APPLICATIONS
OF
RECOMBINAT DNA TECHNOLOGY

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 Pharmaceutical
companies already are
producing molecules
made by recombinant
DNA to treat human
diseases.
 Recombinant bacteria
are used in the
production of human
growth hormone and
human insulin

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Using recombinant cells mass to produce proteins
– Bacteria
– Yeast
– Mammalian
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 • Growth hormone
• Insulin (Humulin) deficiency
– Hormone required to – Faulty pituitary and
properly process sugars regulation
and fats
– Had to rely on cadaver
– Treat diabetes source
– Now easily produced by – Now easily produced by
bacteria bacteria
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Recombinant Insulin

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Production of Insulin by rDNA Technology

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Subunit Herpes Vaccine

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Genetically modified organisms (GMO)
Use of recombinant plasmids in
agriculture
– plants with genetically desirable
traits
• herbicide or pesticide resistant corn
& soybean
– Decreases chemical insecticide use
– Increases production

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 Genetic Engineering of Plants
 Plants have been bred for millennia to
enhance certain desirable characteristics in
important food crops.
 Transgenic plants.
 Crops have been developed that are better tasting,
stay fresh longer, and are protected from disease and
insect infestations.

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The luciferase gene from a firefly
is transformed into tobacco plant
using the Ti plasmid.

Watering the plant with a solution

of luciferin (the substrate for
firefly luciferase) results in the
generation of light by all plant
tissues.

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 Insect-resistant Tomato plants

The plant on the left contains a gene that encodes a

bacterial protein that is toxic to certain insects that feed on
tomato plants.

The plant on the right is a wild-type plant. Only the plant

on the left is able to grow when exposed to the insects.
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Transgenic animals

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A transgenic
mouse

Mouse on right is
normal; mouse on
left is transgenic
animal expressing
rat Growth hormone

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Farm Animals and “Pharm”
Animals
 Trangenic plants and animals
have genes from other
organisms.

 These transgenic sheep carry

a gene for a human blood
protein
– This protein can help in the
treatment of Cystic fibrosis

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 Efficient Food Processing
• By genetically modifying food producing organisms,
the wait time and quantity of certain food processing
necessities are optimized.
• Example: Rennin Production
 The protein rennin is used to coagulate milk in the
production of cheese.
 Now scientists can insert a copy of the rennin gene
into bacteria and then use bacterial cultures to mass
produce rennin.

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 Better Nutrient Composition
 Some plants, during processing, lose some of the vital
nutrients they once possessed.
 Others are grown in nutrient poor areas.
 Both these problems can be solved by introducing genes
into plants to increase the amount or potency of nutrients.
 “Biofortification”
 Example: Golden Rice
 Scientists have engineered "golden rice", which has
received genes from plant and a bacterium that enable it to
make beta-carotene.
 This offers some promise in helping to correct a worldwide
Vitamin A deficiency.

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 Efficient Drug Delivery

• Inserting genes into

plants/animals to
produce essential
medicine or vaccines.
• “Biopharming”

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 Medical biotechnology

• The main reason for medical biotechnology is

to prolong life.
• Other reasons are to ease suffering of palliative
care patients or to increase accessibility for
people with disabilities.

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 Monoclonal Antibodies (mAb)
• They are so called
because they are
clones of an individual
parent cell.
• Antibodies are specific
proteins that target
pathogens invading our
body.
•This technology is used primarily to fight off cancer
cells as these monoclonal antibodies can be “trained” to
target markers that show up on cancer cells.
•The mAbs will then destroy the cancer cell and go
looking for more.
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 Bioprocessing

• Bioprocessing is the mass

production of human
proteins, vaccines, etc…
by genetically modifying
bacteria (or) viruses.
• This allows for a large
quantity of the desired
product to be created in a
short amount of time and
for a relatively low cost.

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 Stem Cells
 Stem cells are introduced
into a damaged area of the
body where, under the
right conditions, will
replace the damaged area

 Often times stem cells are

grown in a lab first to
ensure the right conditions
and then placed into a sick
person.

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 Stem Cells

 The main areas where

stem cells have proven
their worth is in bone
marrow transplants,
replacing damaged heart
tissue after a heart attack
and replacing damaged
nerve tissue which gives
hope to anyone who has
had a.

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 Tissue Engineering
 A form of regenerative
medicine, tissue
engineering is the
creation of human tissue
outside the body for later
replacement.
 Usually occurs on a
tissue scaffold, but can
be grown on/in other
organisms as shown on
the right.

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 SEVERE COMBINED IMMUNODEFICIENCY
(SCID)
• Adenosine deaminase deficiency
• Accumulation of dATP causes the inhibition of
Ribonucleotide reductase which causes B and T
cells unable to divide.

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• SCID is the first genetic disorder successfully
cured by Gene therapy method. 33
 Agriculture Biotechnology
• Produce transgenic plant through plant tissue culture
to improve the crops with the desired traits.
• Resistance develop against abiotic and biotic stress.
• Transgenic plants used as bioreactors for producing
commercial products- protein, vaccines &
biodegradable plastics.

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TECHNIQUE

Agrobacterium tumefaciens

Ti
plasmid

Site where
restriction
enzyme cuts
T DNA
DNA with RESULTS
the gene
of interest

Recombinant
Ti plasmid

Plant with new trait 35

 Environmental Biotechnology
• Environmental biotechnology is the solving of
environmental problems through the application
of biotechnology.
• It is needed to:
– eliminate the hazardous wastes produced by our
other technologies.
– distinguish between similar species and ensure species
are not at risk of extinction.
– to create alternative energy sources (i.e. Biofuels).

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 Biosensors
• A biosensor uses a biological entity (i.e. bacteria) to
monitor levels of certain chemicals (or) uses chemicals
to monitor levels of certain biological entities (i.e.
pathogens).
Current uses of biosensors
includes:
•Detecting levels of toxins in
an ecosystem

•Detecting airborne pathogens

(i.e. anthrax)

•Monitoring blood glucose

levels 37
 Biofuels
 A biofuel is a plant derived
fuel that is deemed more
environmentally friendly
that current fuel sources as
they all release less carbon
dioxide into the atmosphere.
 Ethanol from corn is
placed in many gasoline
varieties in North America.
 Biodiesel is fuel made from
used cooking oil.
 Biogas is made from gases
released by compost or a
landfill. 38