Figure 3

Paralog primer design. (A) Possible primers are identified by finding mismatches within a multiple alignment. Each mismatch is worth one point, which is later used to determine the quality of the primer. (B) Further criteria are: (i) GC-clamp: there should be 1 to 3 G or C bases in the last 5 bases of the primer (8 points); (ii) primers should not have runs of more than 5 identical bases (4 points); (iii) the GC content should be between 40% and 60% (2 points); (iv) the last 5 bases of the primer should not form a secondary structure with the first 5 bases or rather not be the reverse complement (1 points); all primers need to have at least 15 points in order to be considered as candidate.