I have designed a primer which is exactly identical to the target sequence (one of the homologue) but it is giving hit with non target sequence (second homologue) with one base mis-match in between the primer sequence. so i want to know if i will use this primer, will this give two bands (with exact target and with second homologue)
hi naveen,
assuming that you will amplify and sequence with the same primers it takes 2 primers to amplify DNA so it is likely that if the other primer is specific then the amplification will be unique and then there is only one target sequence so the sequencing will work. If the mismatch is at the 3 prime or very close to the 3 prime end of the primer then the primer will not stick well to target dna so there will be no amplification of the wrong product. The quality of your amplimer will benefit from using the highest temperature at which the PCR will work then the effect of the mismatch will be greatest and the yield from it will be the lowest.. If your amplimers share a common reverse primer then if the mismatch is close to the 3 prime end you could try adding DMSO to the pcr reation to try to stop the wrong primer sticking.
If I understand your question correctly, one of the primers in your primer pair have an off-target hit with a single nucleotide mismatch? A single mismatch probably isn't enough to prevent amplification (although as Paul said, a mismatch at the 3' end might be enough to prevent amplification). I would just try to design new primers though, if I were you.
Hi sir,
thank you so much for replying but my question was, i designed a primer to amplify cds but the forward primer showing exact identity to target gene but it also giving hit with its homologue with one base pair in the middle. so i want to know if i will use this primer will it give me two bands as its showing hits with target n the homologue?
In agreement with M N Manoj, Paul Rutland and Spencer Berg above: a mismatch in the middle of a primer is always a candidate for allowing amplification. if you were using an LNA-MGB probe here, it would likely be a different story.
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