Final answer:
Setting an incorrect annealing temperature in the PCR protocol can lead to ineffective primer binding, poor yield, and specificity of the amplified DNA. The incorrect temperature of 65°C instead of the intended 50°C could prevent primers from forming strong Watson-Crick base pairs, thus disrupting the PCR amplification process.
Step-by-step explanation:
Impacts of Incorrect Annealing Temperature in PCR
In the context of Polymerase Chain Reaction (PCR), an incorrect annealing temperature can significantly affect the efficiency and specificity of the amplification process. If the annealing temperature is set too high, in this scenario at 65°C instead of the intended 50°C, the primers may not bind effectively, as they have a lower melting temperature. This suboptimal binding can lead to poor yield and specificity, possibly even resulting in a lack of amplification due to mismatched primer binding or non-specific annealing.
During the PCR technique, the steps of the cycle include denaturation at around 90°C, annealing at approximately 50-60°C, and extension at roughly 72°C. Not adhering to these specified temperatures might compromise the entire PCR experiment. Specifically, in your laboratory error, the higher annealing temperature would hinder the primers' ability to form Watson-Crick base pairs with the template DNA, as their matching sequences would require a lower temperature to successfully pair.
Each PCR cycle is designed to exponentially duplicate the DNA of interest. However, a mismatch in the annealing step, due to an incorrect temperature setting, directly disrupts this duplication process. To resolve such issues, a careful review and adjustment of the thermal cycler settings are required to ensure that the optimum temperature for each phase of the PCR cycle is achieved.