PCR notes for UG students

 What do you meant by PCR? Describe with sketches the mechanism of amplification of DNA by using this method.

Polymerase chain reaction:-

       In 1985-86 a major development was occurred at Cetus Corporation, USA where researchers develop an in vitro method for the amplification of DNA fragment. This method is known as polymerase chain reaction (PCR). The technique becomes important with the discovery of Taq DNA Polymerase by Karymullis 1987. For this discovery and development of polymerase chain reaction, Karymullis shared the Nobel prize for chemistry.

Mechanism of PCR:-

       Polymerase chain reaction is a powerful and widely used technique. It provides a simple method for exponential amplification for specific DNA sequence in vitro. This process require following materials.

(i) Template DNA which is to be amplified.

(ii) 2 nucleoside primers specific to 3' ends of concern DNA segment.

(iii) Amplification buffer containing KCl, Tris-Cl, 1.5mM MgCl₂.

(iv) 4-deoxynucleotide triphosphate i.e. deoxyadinosine triphosphate (dATP), deoxyguanosine triphosphate (dGTP), deoxythymidine triphosphate (dTTP), deoxycytosine triphosphate (dCTP).

(v) A heat stable DNA polymerase, Taq i.e. Thermus aquaticus DNA polymerase Pfu i.e. Pyrococcus furiosus.

Steps of PCR :-

       Action of PCR involves several cycles of amplification however each cycle has 3 steps- denaturation, primer annealing and primer extension (polymerization).

(i) Denaturation:-

       In this step, the template is denatured by raising the temperature to a point where the bonds holding double stranded structure of DNA are disrupted. The denaturation temperature varies between 90-97°c. (usually 94°c). template with high temperature in denaturation of the strand. For efficiency reaction mixture is heated to 94°c for 2 minutes before polymerase, Taq DNA polymerase.

(ii) Primer annealing:-

       Short single stranded DNA molecules are called primers. They are generally between 16-40 basepairs in length with exact match of template sequence is not defined a mixture of primer is used. The perfect matching of primer with the template DNA require correct annealing temperature. The perfect matching primer generally require annealing temperature of 35-60°c. whereas the mixture of primer with some random nucleotide (degenerate primer) require 45-55°c for annealing. Generally primers are added in excess so that the anneal with the template can anneal with each other. The duration of annealing step is usually 1minute.

(iii) Primer extension and chain elongation:-

       This chain extension process carriedout in 72°c. the basic steps of primer extension are as follows-

a) Taq DNA polymerase and deoxyribonucleoside triphosphates are added to the mixture to initiate the synthesis of 2 new chains complementary to the original DNA chains.

b) The DNA polymerase adds nucleotides to the 3' hydroxyl end of the primer and strand growth extens.

       The reaction of chain elongation is exactly similar to the DNA replication that occurs in vivo.

Repeatation of steps:-

       After one cycle of replication, the reaction mixture is heated again to denature the DNA strands. Each DNA strand again binds a complementary primer and the cycle of chain extension is repeated. Typically 20-30 cycles are run during DNA amplification. The duration of each elongation is usually 2 minutes. Each newly synthesized polynucleotide can act a template for the successive cycles.