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Degenerate nucleotides
The IUPAC ambiguity codes used to write primers that cover several related sequences — a variable virus region, a gene family, or a back-translated protein motif — with the degeneracy calculator you need before ordering.
IUPAC code table
| Code | Bases | Meaning | Complement | Fold |
|---|---|---|---|---|
| A | A | Adenine | T | 1 |
| C | C | Cytosine | G | 1 |
| G | G | Guanine | C | 1 |
| T | T | Thymine | A | 1 |
| U | U | Uracil (RNA) | A | 1 |
| R | A, G | puRine | Y | 2 |
| Y | C, T | pYrimidine | R | 2 |
| S | G, C | Strong (3 H-bonds) | S | 2 |
| W | A, T | Weak (2 H-bonds) | W | 2 |
| K | G, T | Keto | M | 2 |
| M | A, C | aMino | K | 2 |
| B | C, G, T | not A (B follows A) | V | 3 |
| D | A, G, T | not C (D follows C) | H | 3 |
| H | A, C, T | not G (H follows G) | D | 3 |
| V | A, C, G | not T/U (V follows U) | B | 3 |
| N | A, C, G, T | aNy base | N | 4 |
| I | inosine | Pairs with A, C, T (and weakly G) | I | 1 |
Degeneracy calculator
Length
14 nt
Total degeneracy
24-fold
3'-terminal 5 bases
3-fold
Degenerate positions
3
Reverse complement: TTDATCCANCCRTC
- Position 3: Y = C, T
- Position 6: N = A, C, G, T
- Position 12: H = A, C, T
Practical guidance
- Keep total degeneracy below roughly 100–150 fold. Each extra fold dilutes the primer that actually matches your template.
- Keep the last 3–4 bases at the 3' end fully defined wherever you can — a mismatch or ambiguity there costs far more than one in the middle.
- Prefer inosine (I) over N in long stretches: it pairs with A, C and T without multiplying the number of species in the tube.
- Raise primer concentration (0.5–1 µM) and use a touchdown program; degenerate pools anneal less efficiently than a matched pair.
- Anchor on methionine and tryptophan codons when back-translating protein — they are the only single-codon amino acids.
Codon redundancy
Met (M), Trp (W)
1 codon — Ideal anchors — no degeneracy at all.
Cys, Asp, Glu, Phe, His, Lys, Asn, Gln, Tyr
2 codons — Third position is a 2-fold code (R, Y).
Ile
3 codons — ATH — third position H (A, C, T).
Ala, Gly, Pro, Thr, Val
4 codons — Third position N; use the first two bases only.
Leu, Arg, Ser
6 codons — Avoid these in degenerate primer regions where possible.
Reference conventions follow the IUPAC-IUB nucleotide ambiguity codes.