BIOTECH 5

 Amplification of Gene of Interest using PCR

• PCR stands for Polymerase Chain Reaction.
• Multiple copies of the gene of interest is synthesized in vitro using two sets of primers and the enzyme DNA polymerase.
• Primers are small chemically synthesized oligonucleotides that are complementary to the regions of DNA.
• PCR includes three major steps-

1. Denaturation

2. Annealing

3. Extension

• Denaturation is the process of heating of target DNA at 94oC to seperate the two strands of DNA.
• Annealing is the process of pairing of primers with complimentary base sequences of the two separated strands.
• Extension is the process of adding complimentary deoxyribonucleotides one by one to the 3/ OH ends of primers by the activity of DNA polymerase and as a result new DNA strand is synthesized.
• If the process of replication of DNA is repeated many times, the segment of DNA can be amplified to approximately billion times by the use of a thermostable DNA polymerase isolated from a bacterium, Thermus aquaticus.
• The amplified fragment can be used to ligate with a vector for further cloning.

Insertion of Recombinant DNA into the Host Cell/Organism

• Recipient cells after making them ‘competent’ to receive, take up DNA present in its surrounding.
• If a recombinant DNA bearing gene for resistance to an antibiotic is transferred into E. coli cells, the host cells become transformed into ampicillin-resistant cell.

Obtaining the Foreign Gene Product

• The foreign gene when gets expressed under appropriate conditions, produces desirable proteins.
• If any protein encoding gene is expressed in a heterologous host, is called a recombinant protein.
• The cells harboring cloned genes of interest may be grown on a small scale in the laboratory or on a large scale in a bioreactor.

Downstream Processing

• Downstream processing is the separation and purification of the product.
• The product has to be formulated with suitable preservatives and the formulation has to undergo thorough clinical trials.

BIOREACTORS

• Bioreactor is the cylindrical vessel in which biological processes is carried out on a large scale.
• The recombinant cells can be multiplied in a continuous culture system wherein the used medium is drained out from one side while fresh medium is added from the other to maintain the cells.
• Bioreactors vessels in which raw materials are biologically converted into specific products, individual enzymes, etc., using microbial plant, animal or human cells.
• A bioreactor provides the optimal conditions for achieving the desired product by providing optimum growth conditions such as temperature, pH, substrate, salts, vitamins, oxygen.
• Bioreactors are of two types-

1. Simple stirred tank bioreactor

2. Sparged stirred-tank bioreactor

• A stirred-tank reactor is usually cylindrical or with a curved base to facilitate the mixing of the reactor contents and the stirrer facilitates even mixing and oxygen availability throughout the bioreactor.
• In sparged stirred-tank bioreactor sterile air is sparged through the reactor.
• The bioreactor has an agitator system, an oxygen delivery system and a foam control system, a temperature control system, pH control system and sampling ports so that small volumes of the culture can be withdrawn periodically.