PCR is carried out in the following three steps :-

Denaturation :-

• The double stranded DNA is denatured by subjecting it to high temperature of 95 degree celsius for 15 seconds.
• Each separated single stranded strand now acts as template for DNA synthesis.

Annealing –

• Two sets of primers are added which anneal to the ‘3’ end of each separated strand.
• Primers act as initiators of replication.

Extension :-

• DNA polymerase extends the primers by adding nucleotides complementary to the template provide in the reaction.
• A thermostable DNA polymerase ( taq polymerase ) is used in the reaction which can tolerate the high temperature of the reaction.
• All these steps are repeated many times to obtain several copies of desired DNA.

Ligation of DNA fragment into a vector

• The vector DNA and source DNA are with the same endonuclease to obtain sticky ends.
• These are then ligated by mixing vector DNA gene of interest and enzyme DNA ligase to form a recombinant DNA.

Insertion of recombinant DNA into the host cell / organism

• Introduction of ligated DNA into recipient cells occurs by several methods, before which the recipient cells are made competent to receive the DNA.
• If recombinant DNA carrying antibiotic resistance ( e.g.ampicillin ) is transferred into E.coli cells, the host cell is transformed into ampicillin -resistant cells.
• On growing transformed cell on agar containing ampicillin, only transformants will grow and others will die.

Culturing the host cells

• The transformed host cells are grown in appropriate nutrient medium at optimal conditions.
• The DNA gets multiplied and expresses itself form desired product.

Extraction of desired gene product –

• When a protein encoding gene is expressed in a heterologous host, it is called a recombinant protein.
• The cells having genes of interest can be grown on a small scale or on a large scale.
• On small scale, the cells are grown on cultures in laboratory and then they expressed protein is extracted and purified by different separation techniques.
• On large scale, the cell are grown in a continuous culture system in which fresh medium is added from one side to medium cells in exponential growth phase and desired protein is collected from other side.
• In large scale method, larger biomass is produced which lads to high yield.

Downstream processing

All the processes to which a product is subjected to before being marketed as a finished product are called downstream processing.

It includes :-

• Separation of the product from the reactor.
• Purification of the product.
• Formulation of the product with suitable preservatives.
• Quality control testing and clinical trials in case of drugs.

Bioreactors

• Bioreactors are vessels of large volumes (100-1000 litres) in which raw materials are biologically converted into specific products.
• It provided all optimal conditions for achieving the desired product by providing optimal growth conditions like temperature PH,substrate, salt, vitamins,and oxygen.
• Stirred – tank bioreactors are commonly used bioreactors.
• These are cylindrical with curved base to facilitate proper mixing of the contents.

A bioreactor has the following components :-

• An agitator system
• An oxygen delivery system
• Foam controls system
• pH control system
• Samping ports to withdraw cultures periodically

The stirrer mixes the contents and makes oxygen available throughout the bioreactor.
Sparged stirred tank reactor is a stirred type reactor in which air is bubbled.