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Degenerate Primer Generator

Back-translate a short peptide into a degenerate primer written with IUPAC codes, with the degeneracy, the codons covered for each residue and the reverse-primer form.

Formula

degeneracy=∏idi,di=distinct codons written for residue i\text{degeneracy} = \prod_i d_i,\qquad d_i = \text{distinct codons written for residue } i
each residue: the IUPAC codon covering the most of its codons and no codon of another amino acid\text{each residue: the IUPAC codon covering the most of its codons and no codon of another amino acid}
degeneracy\text{degeneracy}
number of distinct sequences in the primer pool

How it works

When only a protein sequence is known, a primer has to allow for every codon that could encode it. Writing each residue as an IUPAC codon (Y for C or T, R for A or G, N for any base) gives a single degenerate sequence that represents all of them.

Methionine and tryptophan have one codon each, so they add no degeneracy; leucine, serine and arginine have six codons, which a single IUPAC codon cannot cover, so four are covered and the page says so. Degeneracy multiplies along the primer, and every sequence in the pool competes for the same polymerase, so low-degeneracy stretches are preferred.

Worked example

The peptide M-N-H-K-V-E-W, forward primer, standard genetic code.

  1. M = ATG, N = AAY, H = CAY, K = AAR, V = GTN, E = GAR, W = TGG.
  2. Degeneracy = 1 × 2 × 2 × 2 × 4 × 2 × 1 = 64.

ATGAAYCAYAARGTNGARTGG, 21 nucleotides, 64 distinct sequences. The reverse primer is its reverse complement, CCAYTCNACYTTRTGRTTCAT.

These are the values the calculator opens with, so you can check its output against this example.

Assumptions

  • Every codon of an amino acid is equally likely, so no codon bias is used to reduce the degeneracy.
  • Serine, leucine and arginine are written with four of their six codons.
  • The peptide is exactly conserved in the target. Real homologs differ, which degenerate primers do not cover.

Common mistakes

  • Choosing a peptide rich in serine, leucine and arginine, where the degeneracy is highest and coverage is partial.
  • Ending the primer in an ambiguous third codon position, where a mismatch at the 3′ end prevents extension.
  • Using the full total primer concentration as though it applied to each sequence. Each is present at that concentration divided by the degeneracy.

Related tools

IUPAC Ambiguity Code ToolDecode ambiguity codes and count the sequences they representOpen calculatorDNA Translation Tool (DNA to Protein)Translate in any of six reading frames with a chosen genetic codeOpen calculatorReverse Complement ToolReverse, complement or reverse-complement a sequenceOpen calculatorOligo and Primer AnalyzerLength, GC, Tm, weight and a design-guideline check for a primerOpen calculator

Related equipment

Service documentation, failure modes and parts for the instruments this calculation is used with.