A widely used method for calculating the annealing temperature for a primer used in PCR is 5 degrees below the melting temperature, Tₘ(°C), which is computed by the equation 81.5+0.41×(%GC)−(675/N), where %GC is the percentage of GC nucleotides in the oligonucleotide and N is the length of the oligonucleotide. Notice from the formula that both the GC content and the length of the oligonucleotide are variables. Assuming you have the following oligonucleotide as a primer,

5′-TTGAAAATATTTCCCATTGCC-3′

compute the annealing temperature for PCR. What is the relationship between  and %GC? Why? (Note: In reality, this computation provides only a starting point for empirical determination of the most useful annealing temperature.) <>

Key Concepts

Melting Temperature (Tₘ)

The melting temperature (Tₘ) of a DNA oligonucleotide is the temperature at which half of the DNA strands are in the double-helix state and half are in the 'melted' single-strand state. It is influenced by the nucleotide composition, particularly the percentage of guanine (G) and cytosine (C) bases, which form three hydrogen bonds compared to the two formed by adenine (A) and thymine (T). A higher GC content typically results in a higher Tₘ due to the increased stability of the double helix.

GC Content

GC content refers to the percentage of guanine (G) and cytosine (C) nucleotides in a DNA sequence. It is a critical factor in determining the stability and melting temperature of DNA strands, as GC pairs form stronger bonds than AT pairs. The formula provided in the question incorporates GC content to calculate Tₘ, highlighting its importance in PCR primer design, where optimal annealing temperatures are crucial for successful amplification.

Annealing Temperature in PCR

The annealing temperature in PCR is the temperature at which primers bind to the target DNA sequence. It is typically set a few degrees below the Tₘ of the primers to ensure specific binding while minimizing non-specific interactions. The relationship between Tₘ and %GC is significant because higher GC content generally leads to a higher Tₘ, thus influencing the optimal annealing temperature for effective primer hybridization during the PCR process.