Question

Suggest and describe a technique to obtain multiple copies of a gene of interest in vitro.

Answer 1

Recombinant DNA can be amplified by the PCR. Several identical copies of it can be synthesised in vitro.

For the PCR, two sets of primers (chemically synthesised oligonucleotides that are complementary to a region of DNA), the enzyme DNA polymerase and deoxynucleotides are added.

The PCR consists of three steps:

• Denaturation: Double helical DNA is denatured at a high temperature. DNA polymerase does not get degraded at this temperature, as the DNA polymerase used in this reaction is thermostable. It is isolated from the thermophilic bacterium Thermus aquaticus (Taq).

• Annealing: It is the step in which primers are annealed to single-stranded DNA templates. Two sets of primers (chemically synthesised small oligonucleotides that are
  complementary to the regions of DNA) are used. The temperature of the reaction mixture is lowered to 50–65 °C for some seconds to allow annealing of the primers. DNA polymerase extends the primer in the 5' to 3' direction.

• Extension: Replication of DNA occurs in vitro.

• This cycle is repeated several times to generate up to one billion identical copies of DNA.