Hello,
I am testing different species-specific primer sets. I have two targets (two species, but same gene target). I am trying to select the best primers -- one that will amplify the target species but will not amplify the non-target species.
I am trying a temperature gradient to see if I can find an annealing temperature that will only amplify the target (primer set designed to amplify species 1 -- amplifies species 1 but does not amplify species 2).
Am I approaching this the right way? Is this how primer optimization for specificity should be done?
I tried a temperature gradient last week and got PCR products with both target and non-target species (also got a lot of primer dimers). In the design phase, the primer sets should have been specific enough (using BLAST to test my primer sequences), but I am still getting amplification when I test the primer sets with non-target species DNA.
I think my temperature gradient was too small (Tm-5oC to Tm). Should I try again with a wider temperature range (Tm-5oC to 70oC)? I'm afraid this will be a waste of time and/or that I'm not on the right track.
Any feedback or suggestions would be very much appreciated. Thank you.
I think you're on the right track, a wider range of temps would be a good next step to try. Also look to see if there might be a few more primer sites to try. Some primer sets just don't give great results, even when you think they should. I always check the primer against the entire genome to make sure there is only one complementary site. Also try the OligoCalc from NW University web site (http://biotools.nubic.northwestern.edu/OligoCalc.html) to screen for possible dimer and hairpin formation.
The 3' end of primers is vital to pcr working so if you only have to distinguish 2 type of sample then look in the online databases for a target sequence where there is at least one base and better 2 bases in the last 3 bases of the 3' end of one primer which differ between the sequences. Then run a gradient pcr and you should find temperatures over a few degrees where one primer set works and the other does not. Ensure that the other reverse primer has a higher annealing temperature than the mismatched primer so that it is the mismatch primer that fails to work first in each case
Thank you Paul Rutland for your response. That's very helpful information for me.
I might need to clarify something though.
Right now, I'm testing with a temperature gradient for the PCR reactions:
- Primer set for species 1 + species 2 template DNA (I expect no bands on gel)
- Primer set for species 1 + species 1 template DNA (I expect a band on gel)
After PCR amplification, when I image the gel, I see a band at the expected length for all reactions.
This confuses me because when I was designing the primers, I tested the primer sequences for specificity with Primer-BLAST and BLASTN. With both searches, I did not get any matches with the non-target species, family, or order. Based on my understanding, I thought the primer sequence was specific enough for target-only amplification.
Am I missing something? Perhaps I have a bigger problem than I thought.
Also, I just checked the sequences, as you suggested, and noticed that in my forward primer, I have 6 bases at the 3' end that are found in the non-target species' sequence. And the other forward primer has 5 bases at the 3' end that are found in the non-target species' sequence. Maybe this is enough to be causing my problems.
I haven't mixed the samples yet, so I am not accounting for competition between the primers.
So far, the PCR reactions are either primer set for species 1 + species 2 template DNA OR primer set for species 1 + species 1 template DNA. This is the approach I'm using, at this stage, to test for efficiency/specificity.
I think that the strong homology at the 3' end is a problem and is unfortunate. Nevertheless you have changes in the primer so running a gradient is still a possible option and should be tried. You can also try a dmso gradient...choose an annealing temperature that works and run samples with 1%, 2%......up to 8% dmso.This makes primer binding more stringent so may cause one primer to fail while the other is still working but while you are doing the gradients design new primers.Primers are cheap and your time is not.
- Badges
- Science method