Question

In gradient PCR, what does setting a temperature of 55 °C with a gradient range of 10 mean?

a. Maintaining a constant temperature of 55 °C throughout PCR
b. Varying the annealing temperature from 45 °C to 65 °C
c. Changing the temperature by 10 °C increments around 55 °C
d. Setting a range for the entire PCR process

How is the gradient PCR program relevant to the annealing temperature set in PCR conditions?
a. It is not relevant; the two parameters are independent
b. The gradient PCR program determines the annealing temperature
c. The PCR conditions program overrides the gradient PCR program
d. Both programs work collaboratively to optimize annealing temperature

Answer 1

In gradient PCR, setting an annealing temperature of 55 °C with a gradient range of 10 means the temperature will vary from 45 °C to 65 °C, helping to optimize the annealing phase of PCR. If the annealing temperature is incorrectly set too high, it can lead to inefficient annealing and reduced yield or failure of the PCR reaction.

Step-by-step explanation:

In gradient PCR, setting a temperature of 55 °C with a gradient range of 10 means that the annealing temperature will vary from 45 °C to 65 °C across different wells or zones of the thermal cycler. This gradient allows for the determination of the optimal annealing temperature for PCR primers, which is a crucial step for successful DNA amplification. Gradient PCR is highly relevant to annealing temperature set in PCR conditions as it allows researchers to find the ideal temperature for primer-DNA binding, ensuring specificity and efficiency of the amplification process.

If the annealing temperature was mistakenly set to 65 °C instead of the intended 50 °C, as in the provided scenario, the primers may not anneal efficiently to the target DNA due to the higher temperature being close to or even exceeding the melting temperature of the primers. This could result in a reduced yield or even failure of the PCR reaction, as fewer primer-template hybrids would form, leading to less DNA being amplified during the extension step.