11. BIOTECHNOLOGY: PRINCIPLES & PROCESSES
PROCESSES OF RECOMBINANT DNA TECHNOLOGY
1. Isolation
of the Genetic Material (DNA)
- Treat the bacterial cells/plant or animal tissue with
enzymes
like lysozyme (bacteria), cellulase (plants), chitinase
(fungus) etc. The cell is broken releasing DNA & other macromolecules (RNA, proteins, polysaccharides & lipids).
-
RNA is removed by treating with ribonuclease.
Proteins are removed by treatment with protease. Other molecules are
removed by appropriate treatments.
-
When chilled ethanol is added, purified DNA
precipitates out as a collection of fine threads in the suspension.
2. Cutting
of DNA at Specific Locations
- Purified DNA is incubated with the restriction
enzyme. As a result, DNA digests. These DNA fragments are separated
by a technique called gel electrophoresis.
- Agarose gel electrophoresis
is employed to check the progression of a restriction enzyme digestion. DNA is
negatively charged. So it moves towards the anode. DNA fragments are separated
according to their size through sieving effect of the agarose gel (a polymer
extracted from sea weeds). The smaller sized fragment moves farther.
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The process is repeated with the vector
DNA also.
- DNA fragments can be seen as bright orange
coloured bands when they are stained with ethidium bromide and exposed
to UV radiation.
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DNA bands are cut out from agarose gel. It
is called elution. The cut-out gene of interest and cut vector
are mixed and ligase is added. It creates recombinant DNA.
3. Amplification of Gene of Interest using PCR
- Polymerase Chain Reaction (PCR) is
the synthesis of multiple copies of the gene of interest in vitro using 2
sets of primers & the enzyme DNA polymerase.
-
Primers are small
chemically synthesized oligonucleotides that are complementary to the regions
of DNA.
Steps of PCR:
· Denaturation:
It is the heating of target DNA (gene of interest) at high temperature (940 C) to separate the strands. Each strands act as template for
DNA synthesis.
· Annealing: It
is the joining of the two primers (at 520
C) at the 3’ end of the
DNA templates.
· Extension: It
is the addition of nucleotides to the primer using a thermostable DNA
polymerase called Taq polymerase. It is isolated from
a bacterium, Thermus aquaticus. It remains active in high
temperature during the denaturation of double stranded DNA.
Through continuous replication, the DNA segment is
amplified up to 1 billion copies.
The amplified fragment can be used to ligate with a
vector for further cloning.
4. Insertion of Recombinant DNA into Host Cell
-
Using any methods, the ligated DNA is introduced
into recipient (host) cell / organism. They take up DNA from its surrounding.
- If a recombinant DNA bearing ampicillin
resistant gene is transferred into E. coli cells, the host cells become
ampicillin-resistant cells.
- If the transformed cells are spread on
agar plates containing ampicillin, only transformants will grow. Untransformed recipient
cells will die.
5. Obtaining
the Foreign Gene Product
-
The aim of recombinant DNA technology is
to produce a desirable protein.
-
If a protein encoding foreign gene is
expressed in a heterologous host, it is called a recombinant protein.
- The cells with foreign genes can be grown
in laboratory. The cultures are used to extract the desired protein and purify it
by using separation techniques.
- The cells can also be multiplied in a continuous
culture system. Here, the used medium is drained out from one side while
fresh medium is added from the other. It maintains the cells more
physiologically active and so produces a larger biomass. It yields more desired
protein.
Bioreactors
- These are the vessels in which raw
materials are biologically converted to specific products, enzymes etc., using
microbial, plant, animal or human cells.
-
Bioreactors are used to produce large
quantities of products. They can process 100-1000 litres of culture.
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A bioreactor provides the optimal growth conditions
(pH,
temperature, substrate, salts, vitamins, oxygen) to get
desired product.
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The most commonly used bioreactors are of
stirring type (stirred-tank bioreactor).
It is usually cylindrical or with a curved
base to facilitate the mixing of the reactor contents. The stirrer facilitates
even mixing and oxygen availability. Alternatively, air can be bubbled through
the reactor.
The bioreactor has
·
An agitator system
·
An oxygen delivery system
·
A foam control system
·
A temperature control system
·
pH control system
· Sampling
ports (for periodic withdrawal of the culture).
6. Downstream
Processing
-
It is a series of processes such as separation
and purification of products after the biosynthetic stage.
- The
product is formulated with suitable preservatives. Such formulation undergoes
thorough clinical trials and strict quality control testing.