Polymerase chain reaction- Mechanism and Application

• The Polymerase chain reaction or PCR is a simple and easy method for the production of multiple number of copies of piece of DNA in vitro. 

• This method synthesised large quantities of DNA fragment without the gene cloning.

• This technique was developed by Kary Mullis in California in 1985. He also share Noble prize for chemistry in 1993. 

• The mechanism and detail of Polymerase chain reaction has been described by Erlich. The Polymerase chain reaction can be understood with the three steps:

Denaturation or Melting of target DNA: 

• The target DNA is heated at about 94 degrees Celsius for 15 second. 

• It separate two complementary strands of DNA. Each separate complementary strand now acts as template.

Annealing of Primers: 

• Two primer that are made up of Oligonucleotide are annealed or fixed at 3' end of each  DNA template. 

• This step is done at low temprature about 68 degrees Celsius for 60 seconds. 

• The primers are attached with both template of DNA through hydrogen bond.

Primer extension: 

• In this step, Deoxynucleoside triphosphate and a thermo stable DNA polymerase are added along with Mg ions. 

• The optimum temperature for this step is about 72 degree Celsius. The DNA Polymerase catalyse the polymerization of primers according to separate DNA template.

• The DNA Polymerase that is needed for extension, must be functional at high temprature therefore term thermostable can be used. 

• For this purpose, Taq Polymerase and vent Polymerase are used which are isolated from thermophilic  bacteria like Thermus aquatics and Thermococcus litoralis.

• After completion of one Cycle, the targeted sequences on both strand are copied and four strand are produced. This cycle may repeat 50 times. 

Remember 👌👌one million copies of target DNA sequence may produced  after the 20 cycle and one billion copies may produced after 30 cycles.

Application of Polymerase chain reaction : 

• This technique has wide range of application in field of molecular biology, medicines and biotechnology.

• It is used to amplified of DNA and RNA. It is used to diagnose of disease like AIDS, Tuberculosis,  Hepatitis and other infectious disease.

• This technique is used to detect genetic Disorders like sickle cell anemia  phenylketonuria and muscular dystrophy.

• It is most applicable in forensic science where it is used in search of criminals through DNA finger printing.

• It is also applied in diagnosis of plant disease. A large number of plant pathogen are also detected by Polymerase chain reaction. 

• It has also been found useful in field of archeology. It has been used to clear the doubt for the Wooly mammoth and Dinosaurs.