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Isolation and Purification Processes in Recombinant DNA Technology:
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- Genetic Material (DNA) isolation in Organisms: In the majority of organisms, deoxyribonucleic acid or DNA is the genetic material.
- Required Pure Form: Essential Steps in DNA Isolation for Precision Cutting: In order to cut the DNA with restriction enzymes, it needs to be in pure form, free from other macromolecules.
- Enzymes Needed: DNA from Cellular Harmony for Purification: To release DNA along with other macromolecules such as RNA, proteins, polysaccharides and lipids, treating the bacterial cells/plant or animal tissue with enzymes such as lysozyme (bacteria), cellulase (plant cells), chitinase (fungus) is required.
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- The RNA can be removed by treatment with ribonuclease whereas proteins can be removed by treatment with protease.
- Other molecules can be removed by appropriate treatments and purified DNA ultimately precipitates out after the addition of chilled ethanol.
- This can be seen as a collection of fine threads in the suspension.
DNA that separates out can be removed by spooling
Notes: Genes are located on long molecules of DNA interwined with proteins such as histones.
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How does Recombinant DNA technology enable precision Gene Manipulation?
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- Restriction enzyme digestions are performed by incubating purified DNA molecules with the restriction enzyme, at the optimal conditions for that specific enzyme.
- Agarose Gel Electrophoresis: It is employed to check the progression of a restriction enzyme digestion.
- DNA Charge and Vector Migration: It is a negatively charged molecule, hence it moves towards the positive electrode (anode).
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- The process is repeated with the vector DNA.
- DNA Joining: Creating Recombinant sequences for Genetic advancements: After having cut the source DNA as well as the vector DNA with a specific restriction enzyme, the cut-out ‘gene of interest’ from the source DNA and the cut vector with space are mixed and ligase is added.
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- This results in the preparation of recombinant DNA.
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Power of Polymerase Chain Reaction in Recombinant DNA:
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- Polymerase Chain Reaction in Gene Synthesis: It refers to a reaction in which multiple copies of the gene (or DNA) of interest are synthesized in vitro using two sets of primers (small chemically synthesised oligonucleotides that are complementary to the regions of DNA) and the enzyme DNA polymerase.
- The enzyme extends the primers using the nucleotides provided in the reaction and the genomic DNA as a template.
- DNA Amplification: Billion Copies through Replication: If the process of replication of DNA is repeated many times, the segment of DNA can be amplified to approximately a billion times, i.e.1 billion copies are made.
- Enzyme Required: Amplifying DNA with Thermus aquaticus Polymerase: Such repeated amplification is achieved by the use of a thermostable DNA polymerase (isolated from a bacterium, Thermus aquaticus), which remains active during the high temperature-induced denaturation of double-stranded DNA.
- Ligation Strategies for Gene Amplification and Cloning: The amplified fragment if desired can now be used to ligate with a vector for further cloning.
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Genetic Transformation: Strategies for Recombinant DNA Insertion:
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- Methods of Genetic Transformation: There are several methods of introducing the ligated DNA into recipient cells.
- Recipient cells, after making themselves ‘competent’ to receive, take up DNA present in its surroundings.
- So, if a recombinant DNA-bearing gene for resistance to an antibiotic (e.g., ampicillin) is transferred into E. coli cells, the host cells become transformed into ampicillin-resistant cells.
- If we spread the transformed cells on agar plates containing ampicillin, only transformants will grow, and untransformed recipient cells will die.
- Selective Marker in Genetic Transformation: Since, due to the ampicillin resistance gene, one is able to select a transformed cell in the presence of ampicillin.
- The ampicillin resistance gene in this case is called a selectable marker.
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Recombinant DNA Technologies for Enhanced Protein Production:
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- Power of Alien DNA Multiplication in Recombinant Technologies: When you insert a piece of alien DNA into a cloning vector and transfer it into a bacterial, plant or animal cell, the alien DNA gets multiplied.
- Path to desirable protein production in Recombinant Technologies: In almost all recombinant technologies, the ultimate aim is to produce a desirable protein.
- Hence, there is a need for the recombinant DNA to be expressed.
- Recombinant Protein in Heterologous Hosts: When a protein-encoding gene is expressed in a heterologous host, it is called a recombinant protein.
- The cells can also be multiplied in a continuous culture system wherein the used medium is drained out from one side while the fresh medium is added from the other to maintain the cells in their physiologically most active log/exponential phase.
- This type of culturing method produces a larger biomass leading to higher yields of desired protein.
- Bioreactors and Optimal Conditions for Large-Scale Recombinant Protein Production: To produce in large quantities, the development of bioreactors, where large volumes (100-1000 litres) of culture can be processed is required.
- Provide Essential Requirements: A bioreactor provides the optimal conditions for achieving the desired product by providing optimum growth conditions (temperature, pH, substrate, salts, vitamins, oxygen).
- A stirred-tank reactor is usually cylindrical or with a curved base to facilitate the mixing of the reactor contents.
Simple stirred-tank bioreactor; (b) Sparged stirred-tank bioreactor through which sterile air bubbles are sparged
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Downstream Processing in Recombinant DNA Technology: Optimal Separation, Purification, and Quality Control:
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- Biopharmaceutical Production for Enhanced Separation and Purification: After completion of the biosynthetic stage, the product goes through a series of processes before it is ready for marketing as a finished product.
- The processes include separation and purification, which are collectively referred to as downstream processing.
- Preservatives formulation in Pharmaceuticals: The product has to be formulated with suitable preservatives.
- Such formulation has to undergo thorough clinical trials as in the case of drugs.
- Quality Control and Downstream processing in Biopharmaceuticals: Strict quality control testing for each product is also required.
- The downstream processing and quality control testing vary from product to product.
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