I'm designing primers for species-specific amplification (inside ITS1-ITS2 region). The problem is that when I blast (GenBank) my primer pair, the Forward primer anneals with some isolates (of the same fungus species that i'm targeting) that have one base polymorfism:
Predicted Tm= 58-61ºC product length = 189 Forward primer 1 TTCATCTCCGACTCGCATGT 20 Template 130 ........G........... 149
Reverse primer 1 GAATACCAAGGAGCACAAGGT 21 Template 318 ..................... 298
My goal is to amplify only the 100% specific sequences. Would a higher annealing TºC be able to prevent this unspecific binding?
Question 2: The fungus species i'm targeting have isolates with a diversity of polymorfisms (deletions, insertions, one nucleotide substitution,..) and sometimes the predicted amplicon lenght may vary: 189bp, 190bp, 191bp. Will it be problematic for a RT-PCR quantification assay?
Thanks in advance