Hi all,
I am trying to design a qPCR test to distinguish between closely related sequences. I have some regions that vary between the taxa I am trying to identify but they are still ~75-85% identical over the length of the primer regions. Does anyone have experience that would let them say whether I am likely to amplify my taxon of interest and not its close relatives with sequences so similar?
Cheers,
Jessica
There is a lot of literature on this. Try NCBI's Primer3-BLAST webpage- the standard settings should give you a good idea.
In general, mismatches in the 3'-most half of a primer are the most important, and can cause amplification failure. The closer a mismatch is to the 3' end of the amplification primer, the more important. (Probe mismatches are more important in the center). In addition, you need to consider your PCR conditions- the lower your annealing temp, the less stringent.
There is a lot of literature on this. Try NCBI's Primer3-BLAST webpage- the standard settings should give you a good idea.
In general, mismatches in the 3'-most half of a primer are the most important, and can cause amplification failure. The closer a mismatch is to the 3' end of the amplification primer, the more important. (Probe mismatches are more important in the center). In addition, you need to consider your PCR conditions- the lower your annealing temp, the less stringent.
I completely agree with Dr Gibbons. I have experiences in using primers with 1-3 mismatches. I have seen that single mismatches in the last 8-10 nucleotides from the 3' end could be overcome by lowering the Ta and increasing the template and primer concentration. However, mismatch in the last 5 nucleotides from the 3' end are generally not tolerated by the PCR.
best of luck
SD
Thank you both - I would like to only amplify the species with a perfect match so, I will try to find primers with 3' mismatches and play with the temperature and primer concentration to increase template specificity.
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