Describe the formation of recombinant DNA by the action of EcoRI.

OR

Describe the process of amplification of “gene of interest” using PCR technique.

EcoRI is used in molecular biology to cut foreign DNA and vector DNA to form sticky ends. These sticky ends form hydrogen bonds with their complementary counterparts. The segments with the help of DNA ligases are joined to produce recombinant DNA.

OR

•Multiple copies of the gene of interest is synthesized in vitro using two sets of primers and the enzyme DNA polymerase.

•Primers are small chemically synthesized oligonucleotides that are complementary to the regions of DNA.

•PCR includes three major steps-

a) Denaturation - The process of heating of target DNA at 94 degree Celsius to separate the two strands of DNA.

b) Annealing - The process of pairing of primers with complimentary base sequences of the two separated strands.

c) Extension - The process of adding complimentary deoxyribonucleotides one by one to the 3^/OH ends of primers by the activity of DNA polymerase and as a result new DNA strand is synthesized.

•If the process of replication of DNA is repeated many times, the segment of DNA can be amplified to approximately billion times by the use of a thermostable DNA polymerase isolated from a bacterium, Thermus aquaticus.

•The amplified fragment can be used to ligate with a vector for further cloning.