RECOMBINANT DNA

TECHNOLOGY AND
GENETIC ENGINEERING
By laiba waqar
RECOMBINANT DNA TECHNOLOGY

A technique mainly used to change the phenotype of an

organism (host) when a genetically altered vector is
introduced and integrated into the genome of the organism.
So, basically, this process involves the introduction
of a foreign piece of DNA structure into the
genome which contains our gene of interest. This
gene which is introduced is the recombinant gene
and the technique is called the recombinant DNA
technology.
There are multiple steps, tools and
other specific procedures followed
in the recombinant DNA
technology, which is used for
producing artificial DNA to
generate the desired product
HISTORY

• The recombinant DNA technology emerged with the

discovery of restriction enzymes in the year 1968 by
Swiss microbiologist Werner Arber,
Inserting the desired gene into the genome of the host is
not as easy as it sounds. It involves the selection of the
desired gene for administration into the host followed
by a selection of the perfect vector with which the gene
has to be integrated and recombinant DNA
formed.Thus the recombinant DNA has to be
introduced into the host. And at last, it has to be
maintained in the host and carried forward to the
offspring.
TOOLS OF RECOMBINANT DNA
TECHNOLOGY
• The enzymes which include the restriction enzymes help
to cut, the polymerases- help to synthesize and the
ligases- help to bind. The restriction enzymes used in
recombinant DNA technology play a major role in
determining the location at which the desired gene is
inserted into the vector genome. They are two types,
namely Endonucleases and Exonucleases.
WORKING OF RESTRICTION ENZYMES

• The Endonucleases cut within the DNA strand whereas

the Exonucleases remove the nucleotides from the ends
of the strands. The restriction endonucleases are
sequence-specific which are usually palindrome
sequences and cut the DNA at specific points. They
scrutinize the length of DNA and make the cut at the
specific site called the restriction site.
• This gives rise to sticky ends in the sequence.
• The desired genes and the vectors are cut by the same
restriction enzymes to obtain the complementary sticky
notes, thus making the work of the ligases easy to bind
the desired gene to the vector.
THE ROLE OF VECTORS

• They help in carrying and integrating the desired gene.

• These form a very important part of the tools of recombinant DNA
technology as they are the ultimate vehicles that carry forward the desired
gene into the host organism.
• Plasmids and bacteriophages are the most common vectors in recombinant
DNA technology that are used as they have a very high copy number.
The vectors are made up of
• An origin of replication- This is a sequence of
nucleotides from where the replication starts
• A selectable marker – constitute genes which show
resistance to certain antibiotics like ampicillin
• A cloning sites – the sites recognized by the restriction
enzymes where desired DNAs are inserted.
HOST ORGANISM

• Into which the recombinant DNA is introduced. The host

is the ultimate tool of recombinant DNA technology
which takes in the vector engineered with the desired
DNA with the help of the enzymes.
There are a number of ways in which these
recombinant DNAs are inserted into the host,
namely – microinjection, biolistics or gene gun,
alternate cooling and heating, use of calcium
ions, etc.
Process of Recombinant
DNA Technology
The complete process of
recombinant DNA technology
includes multiple steps, maintained
in a specific sequence to generate
the desired product.
Step-1. Isolation of Genetic
Material.
The first and the initial step in
Recombinant DNA technology
is to isolate the desired DNA in
its pure form i.e. free from other
macromolecules.
Step 2: Cutting The gene
at recognition site
restriction enzymes play a major
role in determining the location at
which the desired gene is inserted
into the vector genome. These
reactions are called ‘restriction
enzyme digestions’.
 Step-3. Amplifying the
gene copies through
Polymerase chain reaction
(PCR).
It is a process to amplify a single
copy of DNA into thousands to
millions of copies once the proper
gene of interest has been cut using
restriction enzymes.
 Step-4. Ligation of DNA
Molecules
.In this step of Ligation, the joining
of the two pieces – a cut fragment
of DNA and the vector together
with the help of the enzyme DNA
ligase.
 Step-5. Insertion of Recombinant
DNA Into Host
.In this step, the recombinant DNA is introduced
into a recipient host cell. This process is termed as
Transformation. Once the recombinant DNA is
inserted into the host cell, it gets multiplied and is
expressed in the form of the manufactured protein
under optimal conditions.
 Applications of
Recombinant DNA
Technology and genetic
engineering
• For the production of vaccines
like the hepatitis B vaccine.
• Production of transgenic plants
with improved qualities like
insect and drought resistance and
nutritional enrichment.
• Therapeutic protein production
like insulin.
• Gene therapy in diseases like
cancer, SCID etc.
• Production of transgenic animals
with improved quality of milk
and egg.
Genetic engineering plays an important role in the
medicinal field.
It is used in the production of hormones, vitamins and
antibiotics.
Gene cloning finds its applications in the agricultural
field.
Nitrogen fixation is carried out by
cyanobacteria wherein desired genes can be
used to enhance the productivity of crops
and improvement of health. This practice
reduces the use of fertilizers hence chemical-
free produce is generated