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Tools in recombinant DNA technology
Tools in
recombinant DNA
technology
While reading this article you may grasp the
concept of tools in recombinant DNA
technology.
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Recombinant DNA technology is the
technique of combining distinct genetic
materials (DNA) and introducing them
into host organisms from two different
species or sources to create new genetic
combinations.
Medicine, science, industry, and
agriculture all benefit from these new
combinations.
The task of inserting a gene into the host
genome is not simple.
The appropriate gene must first be
chosen for injection into the host,
followed by a suitable vector for forming
recombinant DNA by integrating the
gene with the vector.
Herbert Boyer and Stanley Cohen used
E.Coli restriction enzymes to introduce
foreign DNA from plasmids into living
organisms, resulting in recombinant DNA.
Tools used in the
recombinant DNA
technology process
1. Restriction Enzymes: They identify the
site in the vector genome where the
desired gene is introduced.
Endonucleases are enzymes that cleave
DNA strands.
Exonucleases are enzymes that remove
nucleotides from the end of a DNA
strand.
2. Enzyme Ligase: This enzyme joins two
fragments together. The ligase
enzyme is used to join the sticky ends
of the desired gene with the vector.
3. Vector: A plastid plasmid is typically
used to carry and integrate the
desired gene.
4. Host: A capable host cell into which
the recombinant DNA is implanted.
Steps in the DNA
Technology Process
1. Genetic Material Isolation
Isolation of required DNA in its purest
form, that is, without the presence of
other macromolecules.
Within the cell membrane of a typical
cell, DNA coexists with other
macromolecules such as proteins, RNA,
and polysaccharides.
It must be purified and separated from
other macromolecules using enzymes
such as cellulose, Lysozyme, Chitinase,
proteases, and ribonuclease.
The addition of ethanol causes DNA to
precipitate out as a fine thread, and
refined DNA is pulled out, a process
known as spooling.
2. Enzyme Digestion with
Restriction
Restriction enzyme digestion is a
procedure in which restriction enzymes,
which serve as ‘Molecular Scissors,’ cut
DNA at a specific spot.
The purified DNA is subsequently treated
with the restriction enzyme of choice
under optimal conditions for that
enzyme.
Agarose gel electrophoresis is a
technique that uses an Agarose gel to
separate the DNA.
With the assistance of current, Because
DNA is negatively charged, it flows to the
positive electrode and is segregated
based on its size.
This makes it possible to isolate and clip
out digested DNA pieces. Vector DNA
can also be utilised with the same
technique.
3. PCR-based amplification
The polymerase chain reaction (PCR) is a
method for making numerous copies of a
DNA sequence in vitro using the enzyme
DNA polymerase.
By amplifying a single copy of DNA with
the help of PCR, millions of copies of
DNA can be created.
Thermal cyclers require the following
components to execute PCR reactions.
The template is the DNA that will be
amplified.
Primers are short oligonucleotides that
are chemically produced and
complementary to a certain DNA
sequence.
Enzyme: DNA polymerase is a protein
that breaks down DNA. Nucleotides are
required for the enzyme to extend
primers. The cut DNA fragments can be
amplified with PCR and then ligated with
cut vectors.
DNA Recombinant
Host Insertion
In this step, there is a metamorphosis. It
is a procedure in which recombinant
DNA is introduced into a recipient cell,
usually a bacterial cell that has been
rendered competent to accept additional
DNA.
Ca+2 ion therapy, thermal shock,
electroporation, and other procedures
are utilised to make a cell competent. 6.
Obtaining and Culturing Foreign Gene
Products A bit of alien DNA is introduced
into a cloning vector, and the alien DNA
is duplicated in a bacterial cell.
The ultimate goal is to produce desired
protein expression. The protein encoded
by the encoded gene and expressed in
the heterologous host is referred to as
recombinant protein.
To create a big amount of recombinant
protein that benefits humans, a high
volume or cell culture is required.
Bioreactors are the vessels used to do
this. Bioreactors are enormous containers
that can process anywhere from 100 to
1000 litres of cell culture.
A bioreactor provides optimal conditions,
such as pH and temperature, for
biologically converting raw materials into
specialised enzymes and proteins.
Processing in the
Aftermarket
This procedure includes marketing
protein as a finished product following
quality control, purification, and clinical
testing.
Conclusion
Recombinant DNA technology is the
technique of combining distinct genetic
materials (DNA) and introducing them
into host organisms from two different
species or sources to create new genetic
combinations.
Medicine, science, industry, and
agriculture all benefit from these new
combinations.
The task of inserting a gene into the host
genome is not simple.
Frequently asked
questions
Get answers to the most common queries
related to the NEET UG Examination
Preparation.
What is the Recombinant DNA
Technology Principle?
Ans. Ans : With the use of a genetically
modified vector, recombinant DNA
technology alters ...Read full
When and how was this
technique discovered?
Ans. In the year 1869, a Swiss researcher
named Friedrich Miescher discovered the
DNA structure by ...Read full
What role does recombinant
DNA technology play?
Ans. Genetic engineering has shown to be a
very helpful means of reforming and
constructing an enh...Read full
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