Hi all,

I design the AS-PCR assay to genotype the SNP, plasmids were used to guarantee the three cluters. However, it is strange that the hetorozygous plasmid went to wild-type area, while the other two ones stayed where they should be.

I guess it should be due to the specificity of the AS primers, though the tm of the two primers were nealy the same. Therefore, I introduced a mismatch in second, third base near the 3' end. But the hetorozygous site still stay in wild-type cluster if the destabilization strength was weak or directly went to mutant cluster if strong.

If there is anyone know how to deal with this?