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Question
Answer the following question:
Describe the process of amplification of the "gene of interest" using the PCR technique.
Solution
To amplify the gene segment of the 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-degree Celsius). 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 bacteria, 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 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.
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| Enzyme Taq polymerase, is extracted from a eubacterial microorganism Thermus aquaticus from Yellowstone National Park in Montana, USA and isolated by Chien et al. (1976). Taq polymerase successfully replaced the DNA polymerase from E.coli that was being used in PCR earlier and this shift revolutionised the PCR technique. |
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