To begin, I do know that
Tm = 4(G+C)+2(A+T)
So I don't need that to be reiterated. what I wonder is whether wobbles e.g.
R = A/G
Y = C/T
K = G/T
M = A/C
W = A/T
S = C/G
B = C/G/T
D = A/G/T
H = A/C/T
V = A/C/G
N = A/C/G/T
I = Inosin
have a specific melting temperature or do we take all but S and W to always be variable between 2c and 4c, therefore leaving us with a wide range of temperatures to test?
Surely each must have a specific Mt of its own as each has a synthetic structure of its own.
You are mixing up a few different things in your question. Let me begin by saying that the equation Tm = 4(G+C)+2(A+T) is a very vey crude approximation of determining melting temperature and does not account for the negative effect of mismatches nor does it account for length. If you had an oligo 50nt in length the predicted Tm would be above the boiling temp for water. There are much better equations to use that take all those into account, check the IDT web site for example.
Secondly with the exception of adding Inosine, the other combinations are not changing the melting temp. When you design an oligo with R for example, they incorporate approx equal mixtures of both A and G. So 1/2 of the oligos have an A and 1/2 have a G. There is no such base as R. So there would be a population of oligos that bind at the normal melting temp predicted by having the correct base. When you have 2 wobble positions then you would have 25% of the oligo population binding at the normal temperature etc. The only problem with using this "wobble" approach is that when you have multiple N or wobbles, so for example if you had 4 N's in your oligo then only about 1/256 oligo molecules have the correct sequence. So you start running into difficulties when the population size gets too large.
You are mixing up a few different things in your question. Let me begin by saying that the equation Tm = 4(G+C)+2(A+T) is a very vey crude approximation of determining melting temperature and does not account for the negative effect of mismatches nor does it account for length. If you had an oligo 50nt in length the predicted Tm would be above the boiling temp for water. There are much better equations to use that take all those into account, check the IDT web site for example.
Secondly with the exception of adding Inosine, the other combinations are not changing the melting temp. When you design an oligo with R for example, they incorporate approx equal mixtures of both A and G. So 1/2 of the oligos have an A and 1/2 have a G. There is no such base as R. So there would be a population of oligos that bind at the normal melting temp predicted by having the correct base. When you have 2 wobble positions then you would have 25% of the oligo population binding at the normal temperature etc. The only problem with using this "wobble" approach is that when you have multiple N or wobbles, so for example if you had 4 N's in your oligo then only about 1/256 oligo molecules have the correct sequence. So you start running into difficulties when the population size gets too large.
Hi Andrew,
Tm = 4(G+C)+2(A+T) is a traditional way to calculate the melting temperature of an oligo. I usually use the following:
Tm (ºC) = 4(number of G and C) + 2(number of A and T) - 2
anyway, you have online free softwares to introduce your oligo sequences and obtain the Tm, like "oligo properties calculator".
Concerning to the list of codes you show, this is the IUPAC code for degenerative positions in any sequence. E.g. Y means that in this position may be a C or a T, but not a pair CT.
If you want to traditionally calculate the melting temperature of your oligo using the above equation because you have wooble pairs (in RNA usually GU and less abundant CA), I am not sure, but I thing that (G+C)x4 and (A+T)x2 is due to the number of hidrogen bounds the have in Watson-Crick pairs. GC has three and AT has two, and woobles AC and GU have also two, so I would consider wooble pairs as AT pairs.
Good luck!
Kiko.
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