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GC Content Calculator

GC content is the percentage of G and C bases in a DNA sequence: GC% = (G + C) / total bases × 100. Paste a sequence and this calculator returns GC%, AT%, the exact base counts, the length, the GC:AT ratio and a low / typical / high call. It handles RNA (U read as a weak A/T-like base) and IUPAC degenerate codes honestly — S counts as GC, W counts as AT, and every other ambiguous code is reported separately rather than guessed, so a sequence full of Ns is never silently called AT-rich.

GC content is the single most-quoted number about a DNA sequence, and it earns that status because it predicts so much. G·C pairs stack more stably than A·T pairs, so GC content drives the melting temperature of a primer or amplicon, the stringency you need to amplify it, the likelihood of secondary structure, and even which organisms a sequence came from — bacterial genomes range from about 25% GC in endosymbionts to over 70% in soil actinomycetes. It is a composition summary, not a stability model, but it is the first thing anyone checks. This tool lives in the DNA & RNA calculator set and pairs directly with the Primer Tm calculator, which turns the composition you measure here into the annealing temperature you set on the cycler.

The arithmetic is trivial — count the Gs and Cs, divide by the length — but the judgement calls are not. What counts as a "G or C" base when the sequence carries IUPAC degeneracy codes? Is S (= G or C) a GC base? Is a stretch of Ns part of the denominator? Different tools answer these differently and therefore report different GC% for the same sequence. This calculator states its rules plainly: S counts as strong (GC), W counts as weak (AT), and every other ambiguous code (R, Y, K, M, B, D, H, V, N) contributes to the length but is reported separately as ambiguous. That makes GC% a defensible floor rather than a guess.

Why does the denominator decision matter? Because a sequencing read with 20% Ns is not 20% AT-rich — it is 20% unknown. Folding those Ns into the AT count would flatter the sequence; dropping them from the length would inflate GC%. Here the Ns stay in the length and are surfaced on their own line, so you can see exactly how much of your GC% rests on called bases and how much is unresolved. For clean primer design the ambiguous count is normally zero and GC% is exact.

The low / typical / high label is calibrated for primer-length oligos, where roughly 40–60% GC is the comfortable design window: below 40% a primer melts too easily, above 60% it tends to form secondary structure and misprime. That is a rule of thumb for short oligos, not a law for whole genomes, and the how to calculate GC content guide explains where the thresholds come from and when to ignore them. If your real question is reaction setup rather than sequence analysis, the laboratory calculators cover the buffer and stock maths underneath.

A, C, G, T, U plus IUPAC degenerate codes (R Y S W K M B D H V N). Spaces and digits are ignored.

GC content61.1111 %
AT content38.8889 %
Ambiguous bases0 %
Length18 nt
GC bases11
AT bases7
GC : AT ratio1.57143
ClassificationHigh
Base counts
BaseCount
G6
C5
A4
T3

How to use the gc content calculator

Paste your sequence into the DNA sequence box. Case does not matter and spaces or the digit groupings some tools print are ignored. A, C, G, T and U are always accepted; IUPAC degenerate codes (R Y S W K M B D H V N) are accepted and classified as described above.

Press Calculate. The primary card is GC%; the cards below give AT%, ambiguous%, length, the exact per-base counts, the GC:AT ratio and the low / typical / high call.

Read the ambiguous count first. If it is zero your GC% is exact; if it is not, treat GC% as a floor and note how many bases are unresolved.

For a PCR primer, check the GC% sits near 40–60% and then carry the same sequence into the Primer Tm calculator to get the melting and annealing temperature that GC content is driving.

For RNA, the U bases are counted as weak (A/T-like), so GC% describes the guanine-plus-cytosine fraction of the transcript exactly as you would expect.

Copy the result with the Copy button to drop GC%, length and base counts into your notes or a primer-order form.

GC Content formula

GC% = strong / length × 100
strong (GC bases) = G + C + S
weak (AT bases) = A + T + U + W
ambiguous = length − strong − weak (R, Y, K, M, B, D, H, V, N)
GC:AT ratio = strong / weak
Degeneracy semantics follow the IUPAC nucleotide codes: S = G or C (counted strong), W = A or T (counted weak). All other degenerate codes contribute to the length denominator and are reported as ambiguous rather than assigned to GC or AT.

Variables and units

SymbolMeaningUnit
GC%Guanine + cytosine as a percentage of total bases%
strongG, C and the S degeneracy codebases
weakA, T, U and the W degeneracy codebases
ambiguousAll other IUPAC degenerate codes (R Y K M B D H V N)bases
lengthTotal bases after cleaning (spaces and digits removed)bases
GC:ATRatio of strong to weak bases—

How gc content is calculated

  1. Clean the sequence: uppercase it and strip spaces and digits.
  2. Count every base with a full composition pass, separating the unambiguous A/C/G/T/U from the IUPAC degeneracy codes.
  3. Sum the strong bases: G + C + S.
  4. Sum the weak bases: A + T + U + W.
  5. Compute ambiguous = length − strong − weak so the degenerate codes that are neither clearly GC nor clearly AT are surfaced on their own.
  6. Divide strong by length and multiply by 100 to get GC%; compute AT% and ambiguous% the same way.
  7. Classify: below 40% is low, above 60% is high, otherwise typical, and report the GC:AT ratio.

Worked laboratory examples

Example 1 — GC content of a real 18-mer primer

Given: sequence 5′-GCGCATGCGCATACGTAG-3′ (18 nt)

  1. Count G: 6, count C: 5, so strong = G + C = 11.
  2. Count A: 4, count T: 3, so weak = A + T = 7 (11 + 7 = 18, no ambiguous bases).
  3. GC% = 11 / 18 × 100 = 61.1%.
  4. GC:AT ratio = 11 / 7 = 1.57; 61.1% sits just above the 60% line, so the call is high.

Result: GC = 61.1%, AT = 38.9%, 18 nt, ratio 1.57, classified high

Example 2 — An AT-rich vs a GC-rich 16-mer

Given: AT-rich 5′-ATAAGCTTAAGCTTAA-3′ · GC-rich 5′-GCGCGCATGCGCGCAT-3′

  1. AT-rich: G = 2, C = 2 → strong = 4; GC% = 4 / 16 × 100 = 25.0% → low.
  2. GC-rich: G = 6, C = 6 → strong = 12; GC% = 12 / 16 × 100 = 75.0% → high.
  3. Both are 16 nt, but the GC-rich one has three times the strong-base count and will melt roughly 25 °C higher — this is why GC content and Tm move together.

Result: 25.0% GC (low) vs 75.0% GC (high) on the same length

Example 3 — Degenerate bases are reported, not guessed

Given: sequence 5′-GCATGCNNNATGCA-3′ (14 characters, three Ns)

  1. Count G = 3, C = 3 → strong = 6.
  2. Count A = 3, T = 2 → weak = 5.
  3. ambiguous = 14 − 6 − 5 = 3 (the three N bases).
  4. GC% = 6 / 14 × 100 = 42.9%, but the three unresolved bases stay in the denominator, so the true GC% is a floor: it could be higher if the Ns resolve to G/C.

Result: GC = 42.9% (floor), ambiguous = 3 of 14 bases

Example 4 — S and W degeneracy codes land on the right side

Given: sequence 5′-GCSATW-3′ (6 characters)

  1. strong = G + C + S = 1 + 1 + 1 = 3 (S means G or C, so it is a GC base).
  2. weak = A + T + W = 1 + 1 + 1 = 3 (W means A or T, so it is an AT base).
  3. ambiguous = 6 − 3 − 3 = 0; every base is classifiable.
  4. GC% = 3 / 6 × 100 = 50.0%, ratio = 1.0.

Result: GC = 50.0%, no ambiguous bases — S counted GC, W counted AT

Want the reasoning behind the numbers? Read How to Calculate GC Content.

How to interpret the result

A GC% near 40–60% is the comfortable design window for a PCR primer: enough stacking to bind specifically, not so much that the oligo folds on itself or refuses to melt. Outside that band the sequence is not automatically unusable — it is a flag to check secondary structure and to widen your annealing gradient. Carry the number into the Primer Tm calculator to see the melting temperature it actually produces.

The ambiguous% is the honesty metric. If it is zero your GC% is exact and you can quote it. If it is not, GC% is a lower bound and you should say so, because the unresolved bases could be G/C. A high ambiguous% usually means a low-quality read or a consensus of divergent sequences, and no GC figure should be trusted until it is resolved.

The GC:AT ratio is the same information as GC% in a different shape, and it is handy when you compare two sequences of different length: a ratio above 1 means more GC than AT regardless of how long the sequence is. Ratio = 1.0 is exactly 50% GC.

GC content explains a lot but predicts nothing on its own. Two sequences at 55% GC can have very different Tm if their GC bases are clustered rather than spread, because nearest-neighbour stacking depends on order. That is the gap between a composition count (here) and a thermodynamic model (the Primer Tm calculator), and it is the reason both tools exist side by side.

Assumptions

  • The input is a linear nucleotide sequence read 5′→3′; secondary structure, methylation and modifications are not considered.
  • Degeneracy semantics follow the IUPAC nucleotide codes, with S treated as GC, W treated as AT, and all other codes treated as ambiguous.
  • Ambiguous bases remain in the length denominator, so GC% is exact only when the ambiguous count is zero.
  • The low / typical / high label is calibrated for primer-length oligos, not for whole genomes or long constructs.

Limitations

  • GC content is a composition summary, not a stability prediction — it says nothing about base order, stacking or melting behaviour. Use the Primer Tm calculator for that.
  • When degenerate bases are present GC% is a floor, not an exact value; resolve the Ns to get a true figure.
  • The classification thresholds (40% / 60%) are primer-design rules of thumb and do not transfer to genome-scale GC content, where 25–75% is all normal.
  • Base-count order effects — GC clamps, runs and clusters — are invisible to a composition count even though they change how a sequence behaves.

Common mistakes

  • Reading GC% as a melting temperature. They correlate but are not the same; a 55% GC primer can have a very different Tm from another 55% GC primer.
  • Letting a tool silently count Ns as AT, which makes a low-quality read look artificially AT-rich.
  • Applying the 40–60% primer window to a whole gene or genome and concluding the sequence is "abnormal" when 30% or 70% is perfectly ordinary at that scale.
  • Ignoring base order: a GC clamp at the 3′ end matters for specificity even when overall GC% is mid-range.
  • Comparing GC% between sequences of very different length without checking the ambiguous fraction, so one is exact and the other is a floor.

FAQ

What is GC content?

GC content is the percentage of guanine (G) and cytosine (C) bases in a DNA sequence, computed as (G + C) / total bases × 100. It is a measure of base composition and the first number most people check on a new sequence.

Why does GC content matter for PCR?

G·C pairs stack more stably than A·T pairs, so higher GC content raises the melting and annealing temperature of a primer, increases the risk of secondary structure, and makes an amplicon harder to denature. Most primers are designed to sit in a 40–60% GC window for that reason.

How is GC content calculated?

Count the G and C bases, divide by the total number of bases, and multiply by 100. This calculator also classifies degenerate IUPAC codes: S counts as GC, W counts as AT, and any other ambiguous code is reported separately while staying in the denominator.

What counts as a good GC content for a primer?

Roughly 40–60% is the conventional target for a PCR primer. Below about 40% the primer may bind too weakly; above about 60% it is more prone to hairpins, self-dimers and mispriming. It is a guideline, not a hard cutoff, and the final test is always the gel.

How do you handle N and other degenerate bases?

Bases that are neither clearly G/C nor clearly A/T (R, Y, K, M, B, D, H, V, N) are counted as ambiguous: they stay in the length denominator and are reported on their own line, so GC% becomes a defensible floor rather than a guess. Only S (= G or C) is counted as GC and only W (= A or T) as AT.

Does GC content differ between DNA and RNA?

The concept is the same. In RNA, uracil (U) replaces thymine and is counted as a weak, A/T-like base, so GC% still describes the guanine-plus-cytosine fraction of the transcript.

Is GC content the same as melting temperature?

No. GC content is one input to melting temperature, alongside length, salt, primer concentration and the order in which the bases stack. Use the Primer Tm calculator to convert composition into an actual Tm and annealing temperature.

Scientific Sources

External links open in a new tab and carry rel="nofollow". We cite primary references (NIST, NIH/NCBI, peer-reviewed papers) and established manufacturer technical documentation (NEB, Thermo Fisher, Promega, IDT). Calculated results are estimates; your protocol and manufacturer guidance take precedence.

Last reviewed: 2026-09 · Method version: 1.0