Hey everyone,
I'm trying to design a lot of primers and have been having issues with Primer BLAST and Primer3 displaying results that are generating quite a few off-target products.
However, if i screen each of the resulting primers pairs with Primer BLAST, some of them perfectly align with my target and have no off-target products. But, I can't seem to figure out a way to make the settings more stringent, so that only the best primers will show up.
Does anyone know how to use this program? I know it has a lot of potential!
Thanks,
Aaron
Aaron, question is, what do you mean with, best primers?. Tools tend offer diverse primers so you can chose. Maybe you see it better if you take the diferent primer pairs and perform a multiple alignment vs the group of sequences you expect to have in PCR. At the end you will always have to chose based on caracteristics like primer length, GC percent.. etc
hope this helps
Thanks for the Response, Ernesto.
By "best primers", i mean, if the program generates 10 different primer pairs, all with equal GC content, equal Tm's, and equal length, but only differing in their sequence. When I blast these sequences, they vary largely in the number of off-target products that they generate.
Primer3 (which is the software used by Primer BLAST), ends up missing out on some of the highly specific primer sets generated by the Primer BLAST program, and it seems to have issues generating highly-specific primer sets with the settings I'm currently using. When I tweak things to eliminate primers with off-target products, I don't get any results, even though I already have specific primers generated for the same gene using Primer BLAST.
I know there must be a way to get this working, but it is beyond me at this point. Any suggestions?
Thanks,
Aaron
The number of off-target products vary due to the primer squence. Primer3 and other tools, search for primers by exploring the sequence and countig/discounting points when the primer set acumulate caracteristics like GC%, Tm, or length. But the difference amog tools is the search strategy. It may be metaheuristic or deterministic method. This caracteristics may be in differente priority for differente tools. Also take into acount that there are diferent equations to calculate Primers Tm
This is a optimisation problem in computer science and there exist different aproaches for solvig it, thats why results vary.
You may take all the proposed primer sets by the tools and choose with a criteria based on your specific PCR aplications.
If you are trying to automate the primer-findig process with your personal criteria. Think seriously on developing your own software tool. This is actually what we are doing in java languaje.
Hope this helps. Saludos del norte de México
Thanks, Ernesto. I ended up finding that it helps to run the primers against a mispriming library as a selection-rank criterion. I'm still trying to figure out how to optimize these selection conditions in Primer3, because i occasionally get promiscuous primers, depending on the sequence i'm working with.
I'll have to keep playing around with it, as you suggested.
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