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What do you use to check primer secondary structure? - (Jul/03/2013 )

Hi guys,

I am quite new to QPCR. While I am designing my primers, I came across this problem.

I did a primer blast on NCBI and it gave me the primer sequences Tm, complementarity, product size etc.
Then I copy this set of primer sequence into another online program and it gave me a different Tm value.
I am also wondering what do people use to check for the secondary structure? Preferentially free, website based and user friendly.
I tried Primer 3 before but the layout is a bit confusing.
Also, can anyone tell me what does delta G value tell you and what is a suitable G value range for a primer?

Thanks a lot!

-wlpoiacb- has a tools menu with good tools to analyze primer homo and heterodimers.


Yes, it is a bit confusing at the begining to choose which is the best way to analyze the Tm of your primers. Each program has different algorithms and you can find different Tm for the same primer. For me Primer Blast has always work.
I use oligoanalyzer to analyze the secondary structure. Delta G gives you information about the stability of the secondary structure, unfortunately I cannot tell you a suitable G value range. If someone can give us an idea about this field it will be really useful for better understand the concept.


Primer BLAST uses even different algorithms for Tm calculations than the original primer3, from which it's derived. There are many calculations of Tm, they vary a lot in some cases.

If you reset Table of thermodynamic parameters to Breslauer, Salt correction formula to Shildkraut and Lifson and Concentration of divalent cations and dNTPs to 0, you will get same Tm values as primer3. I personally use primer3, because it allows specification of target sequence and excluded regions, features Primer BLAST doesn't offer.
Both Pimer BLAST and primer3 assess secondary structure of primers, both for single and for a pair. In primer 3 it's the values any and 3' in the output, telling you the score for complementarity within any position and more importantly for the 3' position (that's where it would make dimers). As these are only scores and not deltaG values, you just need to look for a lowest score, you can adjust design to get you only low numbers of complematarity, but the default setting (of primer3) is fine for common primer.

Oligo Analyzer from IDT DNA has a very nice pictures of dimers and stuff, but I personally don't remember what deltaG is still fine and which not, so for me the scores on primer 3 are more relevant. The any value is nut that important for formation of dimers, but tells you that the primers may bind out in the reaction. The 3' value is more important, since the default maximum allowed setting in primer3 is 3, that means primers with 0 are the best in this respect and 2 is quite a high value, but I always try to get 0 for the 3' pair complementarity if possible, even if primer3 don't select it as a best pair (you can increase number of pairs designed by changing Number to return).

You can click all parameters in the primer3 output to see a help for each of them. Primer BLAST gives the same complemantarity values as primer3, just additionally BLASTs the primers to the selected sequence set.