GC content calculator
GC content is the percentage of a DNA sequence's bases that are guanine or cytosine: %GC = (G + C) / length × 100. It predicts duplex stability — GC pairs stack more strongly than AT pairs — and drives primer design targets of roughly 40–60% GC.
Result
GC% = (G + C) / length × 100. Ambiguous IUPAC bases count toward length but not toward G+C.
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Formula
%GC = (G + C) / N × 100
Worked example
Given: 33 nt sequence ATGGCTAGCAAAGGAGAAGAACTTTTCACTGGA
- 1.Count bases: A 12, C 5, G 9, T 7 — G + C = 14.
- 2.%GC = 14 / 33 × 100 = 42.4%.
GC content = 42.4%
How the calculation flows
Units & constants
| Primer design target | 40–60% GC; GC-clamp of 1–2 G/C at the 3′ end |
|---|---|
| E. coli genome | ~50.8% GC |
| S. cerevisiae genome | ~38% GC |
| Human genome | ~41% GC average, 30–60% by isochore |
| Ambiguity codes | Count toward length, not toward G+C |
| Price | Free |
Why does GC content matter for primers?
GC pairs form three hydrogen bonds and stack more favourably than AT pairs, so GC content is the coarsest predictor of how tightly an oligo binds. Primers below ~40% GC melt too readily and need longer sequences to compensate; above ~60%, secondary structure and mispriming risk climb. A G or C at the 3′ end (a 'GC clamp') stabilises the exact position where the polymerase extends.
What does GC content do to PCR and sequencing?
GC-rich templates (> 65%) form stable secondary structures that stall polymerases — the standard remedies are DMSO or betaine additives, higher denaturation temperatures, and specialised polymerase formulations. AT-rich stretches cause polymerase slippage in homopolymer runs. Knowing the number tells you which failure mode to guard against before the first run.
Frequently asked questions
- What is a good GC content for PCR primers?
- 40–60%, with the pair matched and a G or C at the 3′ end if possible. Outside that window, compensate with primer length (low GC) or additives and careful design (high GC).
- How are IUPAC ambiguity codes counted?
- They count toward sequence length but not toward G+C, so a sequence with many N's reports a lower bound on GC content. For exact numbers, resolve the ambiguities first.
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