Guidelines for degenerate primer design and usePCR primer sequences are often deduced from amino acid sequences if the exact nucleotide sequence of their target is unknown. However, because of the degeneracy of the genetic code, the deduced sequences may vary at one or more positions. A common solution in these cases is to use a degenerate primer, which is a mixture of similar primers that have different bases at the variable positions. Using degenerate primers can lead to difficulties optimizing PCR assays: within a degenerate primer mixture only a limited number of primer molecules are complementary to the template; the melting temperature (Tm) of primer sequences may vary significantly; and the sequences of some primers can be complementary to those of others. For these reasons, amplification conditions are required that minimize nonspecific primer–template and primer–primer interactions. The following guidance may help when designing and using degenerate primers.
- Avoid degeneracy in the 3 nucleotides at the 3' end, i.e., if possible use Met- or Trp-encoding triplets at the 3' end
- To increase primer–template binding efficiency, reduce degeneracy by allowing some mismatches between the primer and template, especially towards the 5' end, but not the 3' end
- Try to design primers with less than 4-fold degeneracy at any given position.
- Begin PCR with a primer concentration of 0.2 µM
- In case of poor PCR efficiency, increase primer concentrations in increments of 0.25 µM until satisfactory results are obtained