3. Primer annealing temperature (Ta): Too high Ta will produce insufficient primer-template hybridization resulting in low PCR product yield while too low Ta will lead to non-specific PCR products caused by a high number of base pair mismatches. Since Ta is the function of Tm so can be calculated with respect to melting temperature as given below:
4. GC Content: The number of G's and C's in the primer as a percentage of the total bases should be 40-60%.
5. GC clamp: As GC forms a stronger bond than AT, the number of GC content at the 3' end of the primer should not be more than 3 otherwise it will result in non-specific tight binding at regions where G and C are abundant.
6. Primer secondary structures: Primer secondary structures arise as a result of intra or intermolecular attraction within the primer or with other primers which eventually reduce the yield of amplification as the availability of single stranded primers will be limited for PCR. The various types of primer secondary structures are as follows:
Hairpins: Hairpins are loop structures formed by intramolecular interaction within the primer. Optimally a 3' end hairpin with a ΔG of -2 kcal/mol and an internal hairpin with a ΔG of -3 kcal/mol is tolerated generally.
Dimers: A primer dimer is a structure forming a double-strand like structures which is formed by intermolecular interactions between the two primers. If interaction is formed between two homologous or same sense primers, it is called self-dimer whereas if interaction is formed between two different primers, it is called cross-dimer. Optimally, a 3' end self dimer with a ΔG of -5 kcal/mol and an internal self dimer with a ΔG of -6 kcal/mol is tolerated.
Repeats and runs: Repeats are consecutive occurance of di-nucleotide whereas runs are continuous stretch of
single nucleotide. A maximum number of repeats and runs accepted is 4 di-nucleotide and 4 base pairs respectively.