RECOMBINANT DNA

TECHNOLOGY- DEFINITION,
STEPS, APPLICATIONS
STEPS OF GENETIC
RECOMBINATION TECHNOLOGY
1. ISOLATION OF GENETIC
MATERIAL
2. RESTRICTION ENZYME
DIGESTION
3. AMPLIFICATION USING PCR
4. LIGATION OF DNA MOLECULES
5. INSERTION OF RECOMBINANT
DNA INTO HOST
6. ISOLATION OF RECOMBINANT
CELLS
APPLICATION OF RECOMBINANT
DNA TECHNOLOGY
LIMITATIONS OF RECOMBINANT
DNA TECHNOLOGY
 RECOMBINANT DNA TECHNOLOGY
• joining together of DNA molecules
from two different species that are
inserted into a host organism to
produce new genetic combinations
that are of value to science,
medicine, agriculture, and industry.

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 RECOMBINANT DNA (RDNA)
• general name for a piece of DNA
that has been created by the
combination of at least two
strands.

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• They are DNA molecules formed by
laboratory methods of genetic
recombination (such as molecular
cloning) to bring together genetic
material from multiple sources,
creating sequences that would not
otherwise be found in the genome.
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 RECOMBINANT DNA
• in a living organism was first
achieved in 1973 by Herbert Boyer,
of the University of California at
San Francisco, and Stanley Cohen,
at Stanford University, who used
E. coli restriction enzymes to
insert foreign DNA into plasmids.
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 STEPS OF GENETIC RECOMBINATION
TECHNOLOGY
• Isolation of Genetic Material
• Restriction Enzyme Digestion
• Amplification Using PCR
• Ligation of DNA Molecules
• Insertion of Recombinant DNA Into
Host
• Isolation of Recombinant Cells
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 ISOLATION OF GENETIC MATERIAL
• The first step in rDNA technology
is to isolate the desired DNA in
its pure form i.e. free from other
macromolecules.

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 ISOLATION OF GENETIC MATERIAL
• Since DNA exists within the cell
membrane along with other
macromolecules such as RNA,
polysaccharides, proteins, and
lipids, it must be separated and
purified which involves enzymes such
as lysozymes, cellulase, chitinase,
ribonuclease, proteases etc. 10
 ISOLATION OF GENETIC MATERIAL
• Other macromolecules are removable
with other enzymes or treatments.
Ultimately, the addition of
ethanol causes the DNA to
precipitate out as fine threads.
This is then spooled out to give
purified DNA.
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 RESTRICTION ENZYME DIGESTION
• Restriction enzymes act as
molecular scissors that cut DNA at
specific locations. These
reactions are called ‘restriction
enzyme digestions’.

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 RESTRICTION ENZYME DIGESTION
• They involve the incubation of the
purified DNA with the selected
restriction enzyme, at conditions
optimal for that specific enzyme.
• The technique ‘Agarose Gel
Electrophoresis’ reveals the
progress of the restriction enzyme
digestion. 13
 RESTRICTION ENZYME DIGESTION
• This technique involves running out the
DNA on an agarose gel. On the application
of current, the negatively charged DNA
travels to the positive electrode and is
separated out based on size. This allows
separating and cutting out the digested
DNA fragments.
• The vector DNA is also processed using the
same procedure. 14
 AMPLIFICATION USING PCR
• Polymerase Chain Reaction or PCR is a
method of making multiple copies of a DNA
sequence using the enzyme – DNA
polymerase in vitro.
• It helps to amplify a single copy or a few
copies of DNA into thousands to millions
of copies.
• PCR reactions are run on ‘thermal
cyclers’ using the following components: 15
 AMPLIFICATION USING PCR
• Template – DNA to be amplified
• Primers – small, chemically synthesized
oligonucleotides that are complementary to a
region of the DNA.
• Enzyme – DNA polymerase
• Nucleotides – needed to extend the primers by
the enzyme.
• The cut fragments of DNA can be amplified using
PCR and then ligated with the cut vector.
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 LIGATION OF DNA MOLECULES
• The purified DNA and the vector of
interest are cut with the same restriction
enzyme.
• This gives us the cut fragment of DNA and
the cut vector, that is now open.
• The process of joining these two pieces
together using the enzyme ‘DNA ligase’
is ‘ligation’.
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 LIGATION OF DNA MOLECULES
• The result­ing DNA molecule is a hybrid of
two DNA molecules – the interest molecule
and the vector. In the ter­minology of
genetics this intermixing of dif­ferent DNA
strands is called recombination.
• Hence, this new hybrid DNA molecule is
also called a recombinant DNA molecule and
the technology is referred to as the recom­
binant DNA technology. 18
INSERTION OF RECOMBINANT DNA INTO
HOST
• In this step, the recombinant DNA is
introduced into a recipient host cell
mostly, a bacterial cell. This process is
‘Transformation’.
• Bacterial cells do not accept foreign DNA
easily. Therefore, they are treated to
make them ‘competent’ to accept new DNA.
The processes used may be thermal shock,
Ca++ ion treatment, electroporation etc.19
 ISOLATION OF RECOMBINANT CELLS
• The transformation process generates a
mixed population of transformed and non-
trans- formed host cells.
• The selection process involves filtering
the transformed host cells only.
• For isolation of recombinant cell from
non-recombinant cell, marker gene of
plasmid vector is employed.
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 ISOLATION OF RECOMBINANT CELLS
• For examples, PBR322 plasmid vector
contains different marker gene (Ampicillin
resistant gene and Tetracycline resistant
gene. When pst1 RE is used it knock out
Ampicillin resistant gene from the
plasmid, so that the recombinant cell
become sensitive to Ampicillin.

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 APPLICATION OF RECOMBINANT DNA
TECHNOLOGY
• For examples, PBR322 plasmid vector
contains different marker gene (Ampicillin
resistant gene and Tetracycline resistant
gene. When pst1 RE is used it knock out
Ampicillin resistant gene from the
plasmid, so that the recombinant cell
become sensitive to Ampicillin.

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 APPLICATION OF RECOMBINANT DNA
TECHNOLOGY
• Recombinant DNA is widely used in
biotechnology, medicine and research.
• The most common application of recombinant DNA
is in basic research, in which the technology
is important to most current work in the
biological and biomedical sciences.
• Recombinant DNA is used to identify, map and
sequence genes, and to determine their
function.
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 APPLICATION OF RECOMBINANT DNA
TECHNOLOGY
• Recombinant proteins are widely used as
reagents in laboratory experiments and to
generate antibody probes for examining protein
synthesis within cells and organisms.
• Many additional practical applications of
recombinant DNA are found in industry, food
production, human and veterinary medicine,
agriculture, and bioengineering.

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 APPLICATION OF RECOMBINANT DNA
TECHNOLOGY
• DNA technology is also used to detect the
presence of HIV in a person.
• Application of recombinant DNA technology in
Agriculture – For example, manufacture of Bt-
Cotton to protect the plant against ball worms.
• Application of medicines – Insulin production
by DNA recombinant technology is a classic
example.

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 APPLICATION OF RECOMBINANT DNA
TECHNOLOGY
• Gene Therapy – It is used as an attempt to
correct the gene defects which give rise to
heredity diseases.
• Clinical diagnosis – ELISA is an example where
the application of recombinant DNA is possible.

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 LIMITATIONS OF RECOMBINANT DNA
TECHNOLOGY
• Destruction of native species in the
environment the genetically modified species
are introduced in.
• Resilient plants can theoretically give rise to
resilient weeds which can be difficult to
control.
• Cross contamination and migration of
proprietary DNA between organisms.

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 LIMITATIONS OF RECOMBINANT DNA
TECHNOLOGY
• Recombinant organisms contaminating the natural
environment.
• The recombinant organisms are population of
clones, vulnerable in exact same ways. A single
disease or pest can wipe out the entire
population quickly.
• Creation of superbug is hypothesized.

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 LIMITATIONS OF RECOMBINANT DNA
TECHNOLOGY
• Ethical concern about humans trying to play God
and mess with the nature’s way of selection. It
is exaggerated by the fear of unknown of what all
can be created using the technology and how is it
going to impact the civilization.
• Such a system might lead to people having their
genetic information stolen and used without
permission.
• Many people worry about the safety of modifying
food and medicines using recombinant DNA
technology. 30