Single-base extension / Minisequencing

[StefanieCING]

Hi!! I'm trying to optimize a single-base extension assay for 7 positions using both forward and reverse minisequencing primers for each position. Out of these 14 primers, 4 give a high extra peak together with the expected one. I checked the possibility of carrying the primers from the PCR reaction for target amplification but that wasn't the case. I also increased the annealing temperature of the SNaPShot cycling program up to 10 degrees Celsius (from 50 to 60 degrees) to make the annealing more specific but the extra peak is still there with no signs of reduction. Finally, I ordered the same minisequencing primers from a different company in case there was a problem during the purification stage of the primers from the manufacturers but the extra peak is still present!! Please, if anyone has an idea on what should I do more in order to eliminate the extra peaks I would really appreciate it.