Design arbitrary degenerate primers (with non-binding criteria)

Question

[horea.christ]

I would like to design a number of arbitrary degenerate primers (primers with variable bases, e.g. NGATWGCTSATNGC) for a TAIL-PCR.

I would like to be able to specify the following parameters for the design function:

1. Annealing temperature (ideally 44ºC)
2. Degeneracy (how many variations of the AD primer there are (ideally 128 or 256)
3. Binding frequency on (mouse) genome (ideally 1/3000) - though this may be contingent on the degeneracy, and vice-versa..
4. Non-binding area (of course I would not like any variation of the primer to bind to the known-sequence area in between my specific primers and the unknown area which I want to sequence)

Any ideas of any tools that can help me with the above steps? For Primer design I have used eprimer3 thus far (with invariably satisfactory results) - but it seems to not support the concept of arbitrary degenerate primers.

Ideally, if I had a function which could sort out the first of the above criteria, I could use Biopython and BLAST to script the rest myself. Though the binding frequency would take hours if not a day or two, for BLAST to determine it. It would greatly help if anybody already wrote something more ellaborate for this.

[pld]

Maybe try BLAT instead of BLAST and use GNU parallel to speed up the searches?

Answer 1

[John]

I cannot help you with the answer, but perhaps the question...

The annealing temp is based on the number and type of bond between base pairs - so getting exactly 44C is going to be tricky with degeneracy, if not impossible. This is probably why it didn't make sense to code it into primer3. Also, just in general, 44 is pretty low. I will be surprised if your specific primers are really specific at that temp.

The number of variations will be based on the number of degerate bases you use as your primer sequence. Are you saying that this is totally variable and you dont care what regions of genome you target? If thats the case, its going to be up to the number of degenerate base pairs you put in... to get 256 you'll need 4 degerate nucleotides.

I think point 4 means that you want to make sure this super primer also doesn't bind to a certain region?

To beat this puzzle, I would do the following:

You want an annealing temp of 44, so you're going to need roughly 14 to 15 nucleotides on this primer.

4 are already going to be used by the degenerate bp.

Stick them all on one end of the primer, and solve the simpler question of what 10bp sequence targets the mouse genome with a 1/3000 binding frequency.

[horea.christ]

thank you for your suggestions. I doubt, however, that getting a 1/3000 frequency for the first 10 bases and then sticking 4 extra bases on that would help. That would conserve the frequency only if I use N's, and even then it would increase my Tm.

I was thinking of designing degenerate primers with slightly less ambiguous "bases" such as S, or W, or R - that would also allow me to hone in better on the melting temperature, or design GC clamps, etc. can eprimer3 (automatically) calclate the Tm of degenerate primers, or rather give me a Tm range?

[John]

Yes the 4 Ns would increase your Tm, but I accounted for that when I said 10 bases + 4 for a Tm of 44C.

If you stick to W and S you should be able to just treat them as A or G respectively for most melting temp calculations, and you're right that will be better than an N in terms of temperature specificity.

But theoretical temperature specificity is the last of your worries :P

The melting temperatures given by primer3 are rarely completely wrong - but I always do a temperature gradient when trying out new primers for the first time. When designing primers, I look for secondary structure in my primers, the secondary structure of my amplicons, the "uniqueness" of the part of the genome I'm priming (even if its unique, HOW unique is it), the strength of the binding at the 5' end, and how high in temp and salinity I can go.

Even with the most specific primers in the world on paper, I would be totally unphased if the primers happened to amplify two or more regions of the genomic when I try them out for the first time. And thats without the headaches of degeneracy. With degeneracy your observed/expected binding frequency will get further and further apart.

What I'm trying to say is that with primers its always a bit of a gamble. I wrote some very good tools for increasing your odds at this game a few years ago, but its still to this day not something you can 'solve' insilico with such confidence - particularly with the criteria you have outlined. I would say your biggest problem will be preventing your degenerate primers from binding to themselves... by the time you've fixed that you'll probably just have to accept what you've got :P

Answer 2

[Antonio R. Franco]

I don't exactly understand your needs, but I have designed many degenerated oligos using the CODEHOP criteria (Consensus-Degenerate Hybrid Oligonucleotide Primers) with a lot of success in the cloning orthologs and paralogs genes. The rate of sucess depended upon the degree of conservation of the sequences..

Take a look to the page and the published paper. If you need help, I can give you a hand

[John]

I think that's degenerate as in GCG and GCC both code for Alanine, so you can make primers that work across species or similar genes. (actually, this is totally new to me I've never heard that being done before - pretty cool :) )

What Horea wants I think is a primer that binds all over the place by having nucleotides which will have more than one binding partner.

[horea.christ]

Yes, John is right, by degenerate primers I mean primers containing ambiguous bases. in the IUPAC notation that would be "bases" like N, R, Y, W, S, etc.