By Bishnu Marasini1. Primer Length: It is important because melting temperature (Tm) and specificity partially relevant to primer length. Too long primer is inappropriate due to Tm and secondary structure formation and too short primer may not be specific. Generally 15-30 base long primer (or pair) is suitable.
2. Melting temperature (Tm): Tm of both primers (right and left primers that form a pair) is important while setting the annealing temperature in PCR reaction. Annealing temperature is usually set at 5°C less than the lower Tm (among the Tm of both primers). Generally the difference in Tm of both the primers also can’t be more than 5°C.
We can calculate the arbitrary Tm given by Wallace's rule for short length primer;
Tm = 4(G+C) + 2(A+T)°C
It should be around optimum Tm of template dsDNA and shouldn’t be deviate by more than 5°C.
3. %GC content: Although some genomic sequence have comparatively low GC content, around 50% GC content considered as suitable primer.
4. Self Annealing (SA): Self annealing of primer (forming like hair pin) make unavailable to anneal to template DNA. It can be scored as 1 (for each A-T pair) and 2 (for each G-C pair) when it forms the firmest hairpin structure and this score should be less than 20.
5. Pair Annealing (PA): Same as above (no. 4) when pair of primer anneals each other and it also should be less than 20.
6. Best Primer-pair Selection (total score of all of the above parameter): The final score of primer (or pair of primer) is calculated considering above mentioned parameter and best primer (or pair of primer) has the lowest value and always should be less than 10
score 1 per 10% difference in the optimal GC %
score 1 per °C difference from the optimal Tm
score 1 per 10 units of self annealing
score 1 per 10 units of pair annealing
NCBI’s online software “primer 3” for primer design (http://www.ncbi.nlm.nih.gov/tools/primer-blast/)