Tuesday, July 28, 2020

Biotechnology: Principles and Processes- Notes | Class 12 | Part 3: Processes of Recombinant DNA Technology


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.
-    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.
-    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.
-    A bioreactor provides the optimal growth conditions (pH,
temperature, substrate, salts, vitamins, oxygen) to get desired product.
-    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. 

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