Polymerase Chain Reaction - Practice Questions & MCQ

• The PCR is used to amplify a precise fragment of DNA from a complex mixture of starting material usu­ally termed as template DNA or target DNA or gene of interest.
• A basic PCR set up requires several components and reagents. These components include:
  • DNA template that contains the DNA region (target) to amplify
  • Taq polymerase, a DNA polymerase that is heat resistant, so that it can remain intact during the DNA denaturation process. It is isolated from a bacterium, Thermus aquaticus.
  • Two primers that are complementary to the 3' ends of each of the sense and antisense strand of the DNA target.
  • Deoxynucleoside triphosphates, the building-blocks from which the DNA polymerase synthesizes a new DNA strand.
  • Buffer solution, providing a suitable chemical environment for optimum activity and stability of the DNA polymerase.
  • Bivalent cations, magnesium or manganese ions.

Stages in PCR:

• Denaturation:
  • On raising the temperature to about 95° C for about one minute, the DNA gets denatured and the two strands separate.
• Renaturation or annealing:
  • As the temperature of the mixture is slowly cooled to about 55° C, the primers base pair with the complementary regions flanking target DNA strands. 
  • This process is called renaturation or annealing. 
  • The high concentration of primer ensures annealing between each DNA strand and the primer rather than the two strands of DNA.
• Extension:
  • The initiation of DNA synthesis occurs at the 3′-hydroxyl end of each primer. 
  • The primers are extended by joining the bases complementary to DNA strands.
  • The synthetic process in PCR is quite comparable to the DNA replication of the leading strand.