NEETZoologyBiotechnology - Principles and Processes
A student attempts to perform a Polymerase Chain Reaction (PCR) using normal DNA polymerase isolated from Escherichia coli instead of the standard Taq polymerase. If the standard three-step temperature cycle is applied, what mandatory modification must be made to the protocol for successful DNA amplification to occur over multiple cycles?
Options
- AThe denaturation temperature must be lowered to 37^ C to prevent the enzyme from degrading.
- BFresh DNA polymerase must be added manually before the extension step of every single cycle.
- CThe enzyme must be added only once, immediately after the first denaturation step is completed.
- DFresh primers must be added after every cycle to compensate for the lack of thermostability.
Correct answer
B. Fresh DNA polymerase must be added manually before the extension step of every single cycle.
Step-by-step solution
The standard PCR protocol involves a denaturation step at a very high temperature (approximately 94^ C ). Normal DNA polymerase isolated from Escherichia coli is not thermostable and will denature and lose its catalytic activity at this high temperature. If Escherichia coli DNA polymerase is used, it will be destroyed during the denaturation step of each cycle. Therefore, to achieve amplification over multiple cycles, fresh enzyme must be added manually after denaturation and annealing, right before the extension s