Hi everyone. We want to know how can I remove the non-specific band amplification on this variant. Although the amplification of non-specific band is not as intense as the band that we want, but the non-specific band can be still it can be seen (from my point of view). For the design of the primer, we've done mismatch on -3 from the 3' terminal from the SNPs. So far we've done the gradient annealing temperature for this variant and it still not working well. We have done everything that we have thought about. Attached here is the picture of our result. So we hope that we can get any opinion from this platform.
The possible reasons for non-specific binding:
You have done the best try of increasing annealing temperature. You could try an increasing gradient of dmso up to 8% but I expect the results will look similar to the temperature gradient.
If you sequence the odd band it should tell you where it comes from and will allow you to redesign the primers where the unwanted sequence differs from your sequence.
Primers are cheap you could also try mismatch at n, n-1 and n-2 separately with the current return primer .
If your gene has a pseudogene or is a member of a similar sequence gene family you might redesign your reverse sequence in the intron to increase specificity
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