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 44ºC)
- 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 ellaborate for this.