Design arbitrary degenerate primers (with non-binding criteria)

Question

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:

• Annealing temperature (ideally 44°C)
• Degeneracy (how many variations of the AD primer there are (ideally 128 or 256)
• Binding frequency on (mouse) genome (ideally 1/3000) - though this may be contingent on the degeneracy, and vice-versa.
• 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 elaborate for this.

Answer 1

If you already know the basic sequence i.e. the fixed regions in the primer; for e.g. the known nucleotides in the sequence - NGATWGCTSATNGC, then you can implement your algorithm like this:

• Fix the max length of the primers. Lets say 20nt.
• Generate all combinations of primers (20nt): that is pretty straightforward. You will have
  4^N × 2^{(R+Y+S+W+K+M)} × 3^(B+D+V+H) number of combinations, where each alphabet denotes number of such instances. Chek this site for the nucleotide codes.
• Calculate the Tm of the primer-template pairs. UNAfold has a nucleotide hybridization Tm calculator. You can specify parameters like salt concentration etc. There are other such tools too. These can be run on the commandline.
• Discard all primers have Tm below the acceptable limit.
• For those that exceed the acceptable Tm, keep dropping a base from the 3' until the Tm reaches within the range.
• If primer length goes below 14nt then discard the primer.
• Now with all these selected primers check for parameters like self hybridization, GC clamp etc (OPTIONAL). You can use primer3 for these purposes by running it on the primer_check mode.
• Download the nr/nt BLAST database.
• Adjust BLAST parameters for short nucleotide search: Decrease the word length, increase e-value cutoff etc (See this biostar post and BLAST help).
• BLAST your primers against the database in plus-minus mode.
• Worry only about cases where there is significant alignment at the 3' of the primer as only these can cause extension.

These instructions can be automated using any language or shell script.

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