•  The primers should preferably end on a Guanine and Cytosine (GC) sequence so that it can attach with sufficient strength with template. This increases efficiency of priming due to stronger bonding of G and C bases.

•  Runs of three or more Cytosine (C) or Guanine (G) at the 3'-ends of primers should be avoided. This may promote mispriming i.e non-specific binding to G or C rich sequences in the genome other than the target sequence.

•  As Adenine and Thymine base pairs with a single H-bond so Thymine (T) or Adenine (A) residues should be avoided at the 3' end of primers as this weaken the primer's hold on the template DNA.