Question

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

Explain the steps of amplification of the gene of interest using the PCR technique.

Answer 1

To amplify the gene segment of interest, we should know the sequence of the gene of interest. Primers are designed for amplifying the gene of interest. Two sets of primers (chemically synthesized oligonucleotide stretches) that are complementary to the gene of interest, DNA polymerase enzyme, and deoxynucleotides are added. PCR can then be carried out for its amplification.

PCR consists of 3 steps:

• Denaturation: double-helical DNA is denatured by providing high temperature (95°C). DNA polymerase does not get degraded in such high temperatures. The DNA polymerase used in this reaction is thermostable and 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 are used. The temperature of the reaction mixture is lowered to 50-65°C for some seconds to allow annealing of 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 1 billion identical copies of the DNA.

Answer 2

Using the polymerase chain reaction (PCR) technology, a gene of interest can be amplified in the following steps:

1. Denaturation: To split the double-stranded DNA into single strands, it is heated to a high temperature (94-96°C).
2. Annealing: To enable primers to attach to the complementary sequences on the single-stranded DNA, the temperature is decreased (usually between 50 and 65°C).
3. Extension: The temperature is increased to 72°C, which is the ideal temperature for Taq polymerase, which synthesizes a new DNA strand complementary to the template by adding nucleotides to the 3' end of the primers.
4. Cycling: The target DNA sequence is amplified exponentially by repeating these three processes several times (typically 25-35 cycles).