Question

A researcher was trying to amplify a DNA fragment in vitro using a polymerase chain reaction (PCR). All the required components for the reaction except the Taq DNA polymerase were available. The experimenter decided to use DNA polymerase isolated from E. coli instead of Taq DNA polymerase. The E. coli polymerase was added to the reaction at the extension step of each cycle of the PCR. In this scenario, from the options given below, the optimal temperature to be set in the extension step of the PCR is:

• A. 72°C
• B. 37°C
• C. 25°C
• D. 92°C

Answer 1

Answer: (B)

37°C

Answer 2

The Polymerase Chain Reaction (PCR) typically uses a heat-stable DNA polymerase, such as Taq DNA polymerase, isolated from the thermophilic bacterium Thermus aquaticus. Taq DNA polymerase has an optimal activity temperature of around 72°C for the extension step.

However, the question specifies that DNA polymerase isolated from E. coli is used instead of Taq DNA polymerase. E. coli is a mesophilic bacterium, meaning it thrives at moderate temperatures (around 37°C, which is the body temperature of its host). Enzymes isolated from mesophilic organisms, including E. coli DNA polymerase, are not heat-stable and function optimally at temperatures close to their physiological temperature.

Therefore, the optimal temperature for the extension activity of E. coli DNA polymerase would be approximately 37°C. Using higher temperatures like 72°C or 92°C would denature the E. coli polymerase, rendering it inactive. 25°C is too low for optimal DNA synthesis by E. coli polymerase.

The fact that E. coli polymerase would be denatured during the denaturation step (typically 94-96°C) of subsequent PCR cycles makes it unsuitable for a standard PCR protocol unless added fresh in each cycle. However, the question specifically asks for the optimal temperature for its extension activity, assuming it is active at that step.