PCR Primer Annealing Temperature: GC-rich vs AT-rich Primer

Which primer would have a higher optimum annealing temperature?

Of the two primers, 5'-CGCGGGAGCCCCGGGGA-3' and 5'- ATTGAAAATTCTTATTA-3', which one would have a higher optimum annealing temperature?

A. The AT-rich primer

B. One or the other primer, depending on the buffer conditions

C. The GC-rich primer

D. One or the other primer, depending on template DNA sequences

Answer:

The primer with a higher GC content, in this case, 5'-CGCGGGAGCCCCGGGGA-3', would generally have a higher optimum annealing temperature.

Explanation:

The optimum annealing temperature of a primer in PCR (Polymerase Chain Reaction) is largely dictated by its base composition. GC content contributes more toward the melting temperature (Tm) of a DNA duplex because the GC base pair has three hydrogen bonds, as compared to the two in the AT base pair. As a result, a GC-rich primer like 5'-CGCGGGAGCCCCGGGGA-3' would have a higher optimum annealing temperature than an AT-rich primer like 5'- ATTGAAAATTCTTATTA-3'.

The reason for this is during the PCR process, during the annealing step the temperature is lowered to allow the DNA primers complementary to the ends of the target sequence to anneal to the template strands. The higher the GC content of a primer, the higher the temperature needed for successful annealing, simply because more heat is required to break the additional hydrogen bond in GC pairs as compared to AT pairs.

Therefore, when considering which primer would have a higher optimum annealing temperature, the primer with a higher GC content is the favorable choice.