Hi,
Does my TaqMan probe recognize the gene only if the sequence has 100% coverage with the probe or will I also get signal if there is a SNP or even a few bp difference? And if a few bp are ok are there any consequences, e.g. weaker binding?
Thanks
Aleks
It obviously will depend the melting temperature, sequence and length of your probe, but Taqman probes can tolerate a few bp difference (e.g., 1 base mismatch will likely be barely noticeable, but 3-4 mismatches will result in a decrease of amplification efficiency)
Hello,
As mentioned above, the number and positions of the mismatches and stringency of the assay can define the outcome.
To add to Peter's points, the position of the mismatch is also crucial. 5' mismatches are not as big deal as 3' mismatches (Article The Effect of Primer-Template Mismatches on the Detection an...
). If you have a long probe (i.e >30 bases) and your SNP location is in the middle of the probe, mismatch might not be a problem for template hybridization. If you have a 20 base probe with MGB and the mismatch is at the last bases, that could prevent probe-template binding.