Processes of Recombinant DNA Technology
1. Isolation of the Genetic Material (DNA)
- Treat bacterial cells, plant, or animal tissue with enzymes such as lysozyme (bacteria), cellulase (plants), or chitinase (fungus) to break the cell, releasing DNA and other macromolecules (RNA, proteins, polysaccharides, lipids).
- Remove RNA with ribonuclease, proteins with protease, and other molecules with appropriate treatments.
- Add chilled ethanol to precipitate purified DNA as fine threads in the suspension.
2. Cutting of DNA at Specific Locations
- Incubate purified DNA with a restriction enzyme to produce DNA digests, separated by gel electrophoresis.
- Agarose gel electrophoresis checks restriction enzyme digestion progress. DNA, being negatively charged, moves toward the anode, with smaller fragments moving farther due to the sieving effect of agarose gel (a polymer from seaweed).
- Repeat the process with vector DNA.
- Stain DNA fragments with ethidium bromide to visualize as bright orange bands under UV radiation.
- Cut DNA bands from the agarose gel (elution), mix the gene of interest and cut vector, and add ligase to create recombinant DNA.
3. Amplification of Gene of Interest using PCR
- Polymerase Chain Reaction (PCR) synthesizes multiple copies of the gene of interest in vitro using two sets of primers and the enzyme DNA polymerase.
- Primers are small, chemically synthesized oligonucleotides complementary to DNA regions.
Steps of PCR
- Denaturation: Heat target DNA at 94°C to separate strands, each acting as a template for DNA synthesis.
- Annealing: Join two primers at 52°C to the 3’ end of DNA templates.
- Extension: Add nucleotides to the primer using thermostable Taq polymerase, isolated from Thermus aquaticus, which remains active at high temperatures during denaturation.
- Continuous replication amplifies the DNA segment up to 1 billion copies.
- The amplified fragment can be ligated with a vector for further cloning.
4. Insertion of Recombinant DNA into Host Cell
- Introduce ligated DNA into a recipient (host) cell/organism, which takes up DNA from its surroundings.
- If recombinant DNA with an ampicillin-resistant gene is transferred into E. coli cells, the host becomes ampicillin-resistant.
- Spread transformed cells on agar plates containing ampicillin; only transformants grow, while untransformed cells die.
5. Obtaining the Foreign Gene Product
- The aim is to produce a desirable protein.
- A protein-encoding foreign gene expressed in a heterologous host is called a recombinant protein.
- Grow cells with foreign genes in the laboratory, extract the desired protein, and purify it using separation techniques.
- Use a continuous culture system to multiply cells, where used medium is drained and fresh medium added, maintaining physiologically active cells for larger biomass and more protein.
Bioreactors
- Vessels where raw materials are biologically converted into specific products (e.g., enzymes) using microbial, plant, animal, or human cells.
- Produce large quantities, processing 100–1000 liters of culture.
- Provide optimal growth conditions (pH, temperature, substrate, salts, vitamins, oxygen).
- Commonly used are stirred-tank bioreactors, cylindrical or with a curved base, with a stirrer or air bubbling for mixing and oxygen availability.
- Components include:
- An agitator system.
- An oxygen delivery system.
- A foam control system.
- A temperature control system.
- A pH control system.
- Sampling ports for periodic culture withdrawal.
6. Downstream Processing
- A series of processes for separation and purification of products after the biosynthetic stage.
- The product is formulated with suitable preservatives, undergoes clinical trials, and strict quality control testing.
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