Primer melting temperature (Tm) - All primers in the reaction must have a similar melting temperature (Tm) so they anneal to and dissociate from complementary DNA sequences at approximately the same temperatures, allowing each amplification to proceed at the selected temperature.
- Primer melting temperature (Tm), by definition, is the temperature at which one-half of the DNA duplex will dissociate to become single stranded and indicates the duplex stability.
- Primers with melting temperatures in the range of 52-58°C generally produce the best results. Primers with melting temperatures above 65°C have a tendency for secondary annealing.
- The guanine-cytosine (GC) content of the sequence gives a fair indication of the primer Tm. The formula for primer Tm calculation: Tm = [4(G + C) + 2(A + T)] °C
- E.g., primer composition: GACTGCGTTAGGATTGGC; count the number of G+C and A+T and enter into the formula. Hence, Tm = [4 (10)+ 2 (8)] °C = (40+16) °C = 56°C
GC content - Primers should optimally contain 40-60% GC content.
- The GC content is the number of G's and C's in the primer as a percentage of the total bases.
- The presence of G or C bases within the last five bases from the 3' end of primers, known as the GC clamp, helps promote specific binding at the 3' end due to the stronger bonding of G and C bases. More than 3 G's or C's should be avoided in the last 5 bases at the 3' end of the primer.
- Try to have uniform distribution of G and C nucleotides, as clusters of G’s or C’s can cause non-specific priming.
