PCR Primer Length Calculation

Which primer is longer?

If calculated melting temperatures for Primer 1 with 60% CG content and Primer 2 with 30% CG content are the same, which primer is longer? Briefly describe why.

Answer:

Primer 2 is longer. Despite having less CG content, its length compensates to maintain the same melting temperature as Primer 1, which has higher CG content.

Considering the given PCR primers: Primer 1 with 60% CG content and Primer 2 with 30% CG content, if both have the same calculated melting temperatures, Primer 2 must be longer. The reason behind this is that cytosine-guanine (CG) base pairs create a stronger bond compared to the adenine-thymine (AT) base pairs due to the presence of an extra hydrogen bond. Thus, with greater CG content, it takes more heat energy to break these bonds, resulting in a higher melting temperature. To maintain the same melting temperature with a lower CG content, Primer 2 compensates by being longer.

Therefore, in this scenario, Primer 2 is longer than Primer 1 in order to achieve the same melting temperature despite having less CG content.