JCAOligoTutorial1b-TmClarification

How to analyze the 'secondary structure' of your oligos

When designing oligos, there is some ambiguity about whether you want a high or a low Tm, and exactly what is meant by Tm. First, let's disambiguate the term Tm, because there are 3 equally important but different Tm values for the types of oligos you're designing in these tutorials. Let's consider the oligos in the first tutorial's example:

Construction of KanR Basic Part Bca9128

PCR ca1067F/ca1067R on pSB1AK3-b0015  (1054bp, pcrpdt)
Digest pcrpdt                         (EcoRI/SpeI, 1038+11+5, L, pcrdig)
Digest pSB1A2-I13521                  (EcoRI/SpeI, 2062+946, L, vectdig)
Ligate pcrdig and vectdig             (pSB1A2-Bca9128)
-----------------------------------------
>ca1067F  Forward Biobricking of KanR of pSB1AK3  
ccagtGAATTCgtccTCTAGAgagctgatccttcaactc
>ca1067R  Reverse Biobricking of KanR of pSB1AK3  
gcagtACTAGTtccgtcaagtcagcgtaatg

Specifically, let's focus on oligo ca1067F. So I can deal with all the things I need to explain to you, let's modify that oligo to introduce a point mutation into the annealing region. I've put the mutated base in bold so you see what I did.

 ca1067F2  ccagtGAATTCgtccTCTAGAgagctgatcGttcaactc

Now, this new oligo, ca1067F2, will still work just as well as the original. For this particular part, the oligos we designed in the tutorial anneal far upstream and downstream all the regulatory elements needed to express KanR, so not only will this still be a working construction strategy; it will also be a functional KanR part.

There are 3 Tm values we need to discuss. I'm going to call them the "self-Tm", the "First-Round Tm", and the "Second-Round Tm". Before we dig into those, let's define Tm which is also explained here. In a nutshell, the Tm is the temperature at which half the molecules are in one state, and half are in the other. That may not by physicochemically correct, but it's close, and it's a sufficient way to describe things to explain what's going on. Now, the two states in question are different in the three different Tm's for our oligo, but in each case they correspond to two competing secondary structure forms, and a higher Tm means the structure in question is more stable, and lower means lower.

The Self Tm

The self Tm refers to the equilibrium between a DNA oligo that exists in a fully denatured (no Watson-Crick base pairs) and one that is annealed with either itself or other molecules of its own sequence:

Now, the duplex in the illustration looks like normal DNA duplex as we've discussed before. However, in this case, the oligo is annealing to another of its own kind rather than to some template molecule. For both hairpins and duplexes, these are undesirable states for most molecular biology operations because they compete with the template for binding. So, you want the 'self Tm' (which basically is the Tm for whatever is the most stable self-secondary structure) to be low meaning 'not a particularly stable state'. In practice, other design criteria we use such as balanced GC content, avoiding homopolymeric runs, and so forth have the desirable side-effect of eliminating this type of secondary structure (often, but not always). So, personally, I very rarely check the self-Tm of oligos, and I do not recommend you worry about it unless you cannot make a 'normal' oligo for the task at hand. Under those circumstances, try your best to keep it below 40°C. It is definitely not the case that higher self-Tm values will cause PCR to fail--I've done PCRs successfully with 80°C self-Tm. However, really bad self-Tm is definitely something that can cause a PCR to fail. So, if you have a really long oligo or an oligo with a weird sequence, you might check this.

But what region of your oligo to test? The whole thing? The annealing region? Wait a second and think about it, then read on. The answer is the whole thing, but the reason why is complicated. First of all, all you really care about is secondary structure that occludes the annealing region of your oligo, typically the 3' most 20bp. So, why not just look at those 20bp? Well, because most likely what that 20bp will hairpin with (that can often be easily fixed) is the 5' tail you added or a GC-rich recognition site. Those you'll only catch by looking at the entire sequence's Tm. If the most-stable structure involves a hairpin that is in the annealing region itself, well, you'd catch that one however you run the test.

Tools to check self-Tm You can't actually use ApE (please send me an email if you've found this functionality in there). Back in the day, I would use a program called oligotech. It was a .exe kind of thing. The IDT website has a tool that can do all of the above analyses. The 'Hairpin' and 'Self-Dimer' buttons correspond to the two cases outlined here.