When you do PCR, it’s important to set the annealing temperature according to the specific primers you’re using. Each primer should base pair with a specific place on the template DNA (the primer binding site). In the denaturing step, the high temperature will break hydrogen bonds and separate the template DNA strands from one another, as well as preventing the primers from base pairing with the template. During the annealing step, the temperature must be just cool enough to allow the primers to hydrogen bond to the template.
To determine the appropriate annealing temperature, you must first calculate the primer’s melting temperature. The melting temperature of a primer (Tm) is the temperature at which the hydrogen bonds between the primer and the template will be broken. Below this temperature, the primer will bind to the template; above this temperature, it won’t. Melting temperature depends on the number of hydrogen bonds between the primer and its complimentary sequence on the template; a greater number of hydrogen bonds gives a higher Tm. Thus, the melting temperature is proportional to the length of the primer. Also, G-C base pairs form three hydrogen bonds each, while A-T pairs form only two bonds. Thus, the melting temperature is higher for primers with a large percentage of G and C nucleotides. The melting temperature for a short primer can be estimated with this formula: Tm = 4° x (# of G’s and C’s in the primer) + 2° x (# of A’s and T’s in the primer)
a) What is the melting temperature of each of the primers you designed in the question above?
b) The annealing temperature used in the PCR reaction is typically about 5° below the lowest Tm of your primers. What annealing temperature should you use for a PCR reaction with the primers you listed?