Polymerase chain reaction notes- definition, principal, requirements, mechanism

Introduction of PCR-   The polymerase chain reaction was developed by an American biochemist Kary Mullis in the year 1983. By Using PCR it is possible to generate thousands to millions of copies of a particular section of DNA from a very small amount of DNA

• Definition-

 Polymerase chain reaction (PCR) is a lab  techniques of gene amplification with the help of which identical Copies of desired DNA can be Synthesized within a time of short duration.

• Principle of PCR-   

Principle of PCR is based on the ability of DNA polymerase to synthesize new strand of  DNA Complementary to the offered template strand Because DNA polymerase can add a nucleotides only onto the  preexisting -OH group, at the end of the reaction billions of copies are formed.

• Requirements :- 

1. DNA template: Any Source that contains one or more target DNA molecule to be amplified can be taken as DNA template.
2. Taq polymerase -PCR requires a DNA Polymerase enzyme that makes new Strands of DNA using strands as templates. The DNA polymerase used in PCR is Taq polymerase which is isolated from a thermostable bacterium called Thermus aquaticus. It lives in hot Spring and very stable and is  most  active around  70 degree Celsius.Primers- Taq Polymerase can only make DNA when  it is given a primer.
3.  Primer is a very short sequence of nucleotides which provide a starting point for the synthesis of DNA.
4. Two 15-35 nucleotide long oligonucleotide primers which are complimentary to both the strands of target DNA.
5. All four types of deoxyribonucleotides are required- dATP, dGTP, dCTP, dTTP .

All the above mentioned items are mix together to make a reaction mixture.

• Working mechanism of PCR

The process of PCR involves heating and cooling called thermal cycling which is carried out by machine 

called Thermocycler. 

There are three steps in one amplification cycle:- 

1. Denaturation 

2. Annealing or renaturation 

3. Extension 

1)Denaturation-

Firstly, the reaction mixture is heated at about 95C for one minute as a result of which the hydrogen bond between two DNA strands are breaks and two single strands of DNA are completely separated.

 

After denaturation the reaction mixture is slowly cooled down to 55- 66C temperature which is favorable for primers to bind the single stranded DNA strand by way of hydrogen bonding. This process is also called renaturation. 

3) Synthesis or extension or polymerisation:-

It is the final step of amplification cycle. IN the presence of Mg++ ions and dNTPs. (example - dATP, dGTP, dCTP, dTTP). 

Taq polymerase which is a thermostable bacteria initiates the synthesis of DNA at 3’ hydroxyl end of each primer and The primers are extended by joining the bases complementary to DNA strands. Thus, in the first cycle the target DNA is copied from the primer sites for various distances until the second cycle starts. 

After that second cycle of amplification is started for this the DNA strands are denatured which are original and newly synthesized long templates annealed with primers and subjected to DNA 

synthesis. 

 The no. of DNA strands becomes four times at the end of second cycle. In this way at the end of 32nd cycle of PCR, about million fold target DNA is synthesized. 

 After PCR has been completed the results are usually visualize using gel electrophoresis (A technique in which fragments of DNA are pulled through gel by a electric current and it separates DNA fragment according to size.