I've designed a paired primers to amplify a specific target within CYP2C19. After checking the potential amplicons, we thought we could get a uniq amplicon by using the two primers as followed:
However, after PCR, we did Sanger verification and found that the target is not a uniq band. We then blast the amplicon sequence and get a similar amplicon with the same length, which is considered as the reason for this amplicon mix or multiple bands.
What we did to screen the primers is to window-size the sequence and get the potential paired-primer using the similar parameters as NCBI-Primer-blast.
Is there any method to force the blast program to have more than 4 mismatches while cost a little for penalty, etc., allow 5 mismatches.