Primers Design: Choosing the right primers is one of the most crucial factors that determine the results of the PCR. Primer should be purified, or at least desalted. Crude primers generally do not work well for sequencing.

•      Primer Length of 18-24 bases.

              The specificity is generally controlled by the length of the primer and the annealing temperature of the PCR reaction. the optimal length of PCR primers between 18 and 24 bases tend to be generally accepted, which is suitable for specificity and for primers to bind easily to the template at the annealing temperature.

•      Primer Melting Temperature (Tm) of 50-60°C.

•      40-60% G/C content.

              Primers for PCR and sequencing should have a GC content between 40% and 60%.The GC content (the number of G's and C's in the primer as a percentage of the total  bases) of primer should be 40-60%.

•      Start and end with 1-2 G/C pairs.

              Primer pairs should not have complementary regions.

•       The structure of the primer should be relatively simple and contain no internal secondary structure to avoid internal folding. Presence of the primer secondary structures produced by intermolecular interactions can lead to no yield of the product. They have a bad effect on primer template annealing by reducing the availability of primers to the reaction and thus the amplification.

•       Avoid Cross Homology.

               To improve specificity of the primers, it is necessary to avoid regions of homology. Primers designed for a sequence must not amplify other genes in the mixture.

Primer design is aimed at obtaining a balance between two goals: specificity and efficiency of amplification.