Question

1. How is PCR used to isolate a particular part of the genome?

2. What is the purpose of a PCR primer?

3. Why is PCR used after isolating genomic DNA for the purpose of examining a SNP?

Answer 1

1) PCR or Polymerase Chain Reaction is a technique that is used to amplify genome. After athe isolation of a genome by standard DNA isolation protocols, PCR can be used to amplify a selected gene or our gene of interest. This is done by designing a primer sequence that is complementary to our gene of interest. So, each time as the cycles in PCR repeats, the primer gets annealed complementary to our gene of interest. This is how a particular part of genome is isolated.

2) The 3 steps in a PCR cycle includes,

Denaturation, Annelaing and Extension.

In denaturation step, a temperature of about 94⁰c is provided to melt the hydrogen bonds between the DNA strands. And during annealing pocess, when the temperature is reduced, It is the Primers that get annealed ( Hydrogen bonded) to one side (opposit sides) of each complementary strands. As the polymerase enzyme can only perform addition of nucleotides to an existing free 3' OH ( ie., it cannot start the synthesise of a new strand ) it is the primer that provides a free 3' OH (hydroxyl group).So, that during the extension step the polymerase could synthesize a new strand complementary to the parental strand.

3) SNP or Single Nucleotide Polymorphism can be defined as a single base change in a DNA sequence that occurs in significant proportion in a large population.

SNP can be observed through PCR-RFLP. Here, the genome is digested using restriction endonucleases and the fragments are run on a Gel electrophoresis. So, before perfoming this, PCR should be carried out. It is because the isolated genome as such, the quantity will be loo less. So, to increase the quantity ( amplify) , PCR is done, so that it makes good visualization in an Electrophoresis apparatus.