Question

Many copies of a specific gene of interest are required to study the detailed sequencing of bases in it. Name and explain the process that can help in developing large number of copies of this gene of interest.

 

 

Answer 1

Polymerase chain reaction (PCR) is used for developing multiple copies of DNA containing the gene of interest.
In this process, two sets of primers (chemically synthesised oligonucleotide stretches that are complementary to a region of DNA), enzyme DNA polymerase and deoxynucleotides are added.


PCR consists of three steps:

• Denaturation: It occurs at 94°C in which double helical DNA is denatured and opens into the two pieces of ssDNA. DNA polymerase does not get degraded in such high temperatures, as it is isolated from thermophilic bacteria, Thermus aquaticus (Taq) and is thermostable.

• Annealing: It occurs at 50-65° C for some seconds to allow annealing of primers to single stranded DNA templates. DNA polymerase extends the primer in 5' to 3' direction.

• Extension: It occurs at 72°C in which replication of DNA occurs in vitro.

This cycle is repeated several times to generate up to 1 billion identical copies of the DNA.