I am designing primers and would like to retain as much specificity as possible. However, due to variation in the gene I am coming across many issues where wobbles would need to intervene, and would like to avoid making the assay over sensitive or at risk of primer dimers appearing.
Hi, the standard code letters for specifying a wobble are:
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
Normally you shouldn't use more than 3 wobbles in a primer (but I have seen primers with more wobbles and they are working).
Cheers, Nadine
Hi, the standard code letters for specifying a wobble are:
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
Normally you shouldn't use more than 3 wobbles in a primer (but I have seen primers with more wobbles and they are working).
Cheers, Nadine
Thank you! There are an amazing number of helpful people on research gate!
You can use primers with wobbles for semi-random mutagenesis, e.g. TXY will give you Tyr, Cys, Ser or Phe but never a stop codon or Leu or Trp, GAX would give you only Asp or Glu, or GSX only Gly or Ala. From my own experience I never had problems during the PCR&cloning steps but the clones I got didn't show the expected distribution, either the PCR or the ligation step is more efficient for some of them bases, i.e. instead of an expected 1/3:1/3:1/3 ratio I got something like 5:4:1.
Dear Andrew, you may want to use nucleotide analogues or base modifications or alphabet extenders in order to make your primers insensitive to insignificant polymorphisms (for the specified or future cases). For example, check http://eu.idtdna.com/catalog/Modifications/Modifications.aspx?catid=7 or http://www.sigmaaldrich.com/life-science/custom-oligos/custom-dna/product-lines/modifications.html or whatever vendor's synthesis service you are used to.
Hi Andrew, in addition to inosine, which will pair with A, T, G or C, you can exploit the non-canonical G:T basepair, which lets you use G as a universal purine and T as a universal pyrimidine. These are all the possibilities that I know of. There are a number of other nucleotide analogues that have other specificities, but few of them will work in primers because DNA polymerase cannot read through them, so you get a truncated, uncopyable amplicon. There don't seem to be any programs that will calculate Tm for such primers, so I just use Tm=2AT+4GC, counting G:T as an AT and assume that inosine pairs make no contribution. It worked for me.
Of course you can, and it works fine. We do it regularly for real-time PCR RT-PCR used in diagnosis analysis.
Hi,
Depnds on what exactly You want to do. Do you have several similar genes in one sample or do you want to use the same primers for many different samples?
In general, if it would be enough to have one position with a wildcard, I WOULD PREFER to order and to have 2 primers of a different sequence and to mix them on my own and only when it is necessary.
Andrew, just complementing the first answer by Nadine Gund. If by any chance you are going after three 'woobles' in your primer try to allocate or having them closer to the 5' of the primer. Remember that stable anchoring of the primer at the 3' terminus is critical for Taq to run over the synthesis, so avoid woobles at or closer to the 3' terminus. Cheers!
I agree with Francisco. Keep the 3' end as free from wobbles as possible. However, it is possible to have a G:T basepair just one position from the 3' end and still get good amplification. We've done this and the assay works just fine. But I wouldn't like to make a habit of it.
Thanks for your information. Could you please tell me how does the wobble work in primer annealing? When I use Y in the primer sequence - what is the actual base in that position?
Thanks Dr Andrew for your kind information. If my primer contains 3 wobbles - such as H (A/C/T), B (C/G/T), K (G/T) - Is it possible to make such a mixture with each nucleotide combination - 18 types of sequence variation in forward primer alone (~6% each).
My DNA template is complementary to only one of those primers. Does this give optimal amplification of my target gene sequence? (I presume primer could become deficient for amplification)? Do I need to increase the primer concentration in such situation?.
Hi Ramalingam,
This is the problem with wobbles: a large percentage of the sequences synthesised will be 'sterile' combinations that don't have full homology to anything. Others will show varying degrees of cross-reaction depending on the position and type of the mismatch. As far as I'm aware, there is no software that will accurately calculate the Tm of mismatched primers. Anyone who comes up with a link to one gets every upvote I'm allowed to give!
My advice is to use inosine for H and B, while keeping the K. Inosine is a wild-card nucleotide that will allow any base opposite it. I usually make the assumption that it makes no contribution to stability, so Tm=2(AT) + 4(GC) + 0(I). This reduces the number of primer sequences to two, which is manageable. Unfortunately the K gives two different Tm's, differing by about 2C, but this shouldn't matter too much.
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