Cheuk Tam

If you need to know about secondary structures of your primers, then NetPrimer of http://www.premierbiosoft.com would give you good advice on it.

You can also analyse the hairpin and cross dimer.

Best.

Hi Carolina and Danny,

Thank you so much for your suggestions. I'll have to apologize for a typo in my question. The last word was supposed to be Primer3, not Primer.  I really like the idtdna analyzer, and I'm excited to try out NetPrimer.

One challenge I'm running into is that I'm trying to design primers for many genes and wanted to efficiently filter out primers that are likely to form hairpins or primer dimers.  I've tried going to tools like IDT's analyzer and looking at secondary structures and delta G values for multiple primer candidates for each gene (generated by Primer Express) but was wondering if there was an efficient way to evaluate primer sets for multiple genes.  A friend suggested that I use Batchprimer3 to streamline the process.  One important point was that I needed to set the Max self and Max 3' self complementarity scores properly since that's how the Primer3 algorithm evaluates hairpin and primer dimer formation. Are there good rules of thumb for these parameters?  Are there ways to convert the complementarity scores into delta G values or vice versa?

Thank you so much for your help.

Sincerely,

Cheuk

Hi Carolina,

Thank you for your reply.  I also found some more information on this question, although I encountered another (please see the end).  My friend said her lab essentially uses trial-and-error with Primer3. She starts with the most stringent setting (e.g. 1 and 0) and increments up if there are no results. Her lab uses 2 and 2 as a general cutoff, but if there are no results, she settles for primers with higher complementarity scores. She doesn’t use IDT to check delta G values, but as Francisco in the following post mentions, we can use an mFold-based tool like IDT to double-check for hairpins and dimers. The last step would be to use BLAST or Primer BLAST to check for specificity.

https://www.researchgate.net/post/What_essential_parameters_should_be_checked_for_primer_designing_and_what_software_should_be_used

I guess all the tools we mentioned are necessary. We may have to adjust the stringency in the initial design to narrow down the candidates to check with IDT.

I did come across one other parameter to consider.  The attached article suggests using IDT to check to see if the amplicon forms secondary structures.  Have amplicon secondary structures ever been an issue?  

Thank you again for sharing your experience with primer design. It really helps to hear how theory plays out in real life.