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 procedure followed in the Recombinant DNA technology,
which is used for producing artificial DNA to generate the desired
product. Let’s understand each step more in detail.
What is Recombinant DNA Technology?
The technology used for producing artificial DNA
through the combination of different genetic materials (DNA) from different
sources is referred to as recombinant DNA Technology. Recombinant DNA
technology is popularly known as genetic engineering.
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,
- Endonucleases
- Exonucleases.
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 vectors – 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 nucleotide from where the replication starts, a selectable marker –
constitute genes which show resistance to certain antibiotics like ampicillin;
and 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 the recognition sites.
The 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 the restriction enzymes.
Step-4. Ligation of DNA Molecules.
In this step of Ligation, 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
after the insertion of the recombinant DNA into the host cell, it
gets multiplied and is expressed in the form of the manufactured
protein under optimal conditions.
As mentioned in Tools of recombinant DNA
technology, there are various ways in which this can be achieved. The
effectively transformed cells/organisms carry forward the recombinant gene to
the offspring.
Application of Recombinant DNA Technology
- DNA technology is also used to detect the
presence of HIV in a person.
- 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
- Recombinant DNA technology is widely used in
Agriculture to produce genetically-modified organisms such as Flavr
Savr tomatoes, golden rice rich in proteins, Bt-cotton to protect the
plant against ball worms and lot more.
- In the field of medicines, Recombinant
DNA technology is used for the production of Insulin.
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