Final answer:
Primers in PCR may start having secondary annealing problems if the temperature is much above their melting temperature, which is ideally around 78°C. The optimal annealing temperature is around 55°C, and setting it too high, such as at 65°C, may result in decreased specificity and efficiency of the PCR.
Step-by-step explanation:
In the polymerase chain reaction (PCR) process, primers can begin to experience secondary annealing issues, typically when the temperature is significantly higher than their melting temperature. Primers should be designed to have a melting temperature of approximately 78°C and should anneal to the template DNA at around 55°C, which is usually 5°C below their melting temperature for optimal results. If the annealing temperature is incorrectly set to a higher temperature, such as 65°C instead of the intended 50°C, the primers may not anneal efficiently to their target sequences. This can lead to a decline in PCR specificity and an increase in non-specific products due to the primers binding at mismatched sites.