Question

A biology student is performing PCR for the first time. She adds the genomic DNA obtained after isolation along with the corresponding primers in a test tube. She also adds Taq polymerase and deoxyribonucleotides in the tube. The reaction mixture is kept in a water bath that measures 37℃. But DNA amplification does not occur. Why?

• A. Thermocycler was not used to maintain temperatures required for PCR
• B. DNA primers were degraded at 37℃
• C. DNA polymerase was added instead of RNA polymerase
• D. Not enough DNA was available for the DNA polymerase to act on

Answer 1

The correct option is A Thermocycler was not used to maintain temperatures required for PCR

PCR or polymerase chain reaction is a technique used in molecular biology to create several copies of a certain DNA segment. Even a small amount of DNA is sufficient for carrying out this process.

For PCR a thermocycler is required, which provides the optimum temperatures required for the three different steps in PCR- denaturation, annealing and extension.

Denaturation involves the breaking of hydrogen bonds between the two strands of a double-stranded DNA segment. It requires a very high temperature around 96°C. This separates the two DNA strands and primers can attach to them in the next step which is called annealing.

A temperature between 55 - 65℃ is required for the primers to anneal to specific DNA sequences.

Finally, the addition of dNTPs to primers and extension of complementary strands happens at around 72℃ of temperature, with the help of Taq DNA polymerase.

Hence, by placing the water bath at a constant temperature (37℃), the PCR process did not proceed due to inappropriate temperature conditions. Due to this, DNA amplification did not occur.

RNA polymerase (helps in the synthesis of RNA) is not added during PCR as it is not required.