How this works
GC content is the percentage of bases in a nucleic acid sequence that are either guanine (G) or cytosine (C), as opposed to adenine (A) or thymine/uracil (T/U). It's one of the most basic descriptors of a sequence and shows up in nearly every aspect of molecular biology, because GC and AT base pairs differ in thermal stability: G pairs with C through three hydrogen bonds, while A pairs with T through only two. More GC content means a more stable double helix and a higher melting temperature. Across the tree of life, GC content varies from about 13% in extreme low-GC bacteria to over 75% in some Streptomyces species — and within a single genome it varies by region, with isochores in mammals and CpG islands in vertebrate promoters being two well-studied examples.
The formula is straightforward: GC% = (G + C) / (A + T + G + C) × 100. The calculator above accepts raw sequence or pasted FASTA (with the `>header` line stripped automatically), is case-insensitive, treats U as equivalent to T (so RNA sequences work), and ignores any non-base characters — whitespace, numbers, punctuation, gaps, alignment markers, and IUPAC ambiguity codes (N, Y, R, W, S, K, M, B, D, H, V). The denominator counts only the four canonical bases, so a sequence that's 90% A/T/G/C and 10% N will report GC% based on the 90% you can actually call. If a meaningful fraction of your sequence is N, treat the GC% as approximate and consider running the calculation on the unmasked subset only.
A few practical points. (1) GC content of a primer matters more than the genome's overall GC content for most lab use cases. PCR primers typically aim for 40-60% GC; outside that range you risk poor priming (too low GC means weak binding) or secondary structures and stable mismatches (too high). (2) For full-genome or long-contig analysis, GC content varies meaningfully by window size — a single number for a whole bacterial genome is informative, but for a mammalian chromosome you usually want a sliding-window plot. The number this calculator returns is the simple average across whatever you paste. (3) The melting-temperature implications are real but not directly proportional: a 50% GC primer melts ~5-10 °C higher than a 30% GC primer of the same length, but the exact relationship depends on length, salt concentration and nearest-neighbour context. Use a dedicated Tm calculator (with the nearest-neighbour method) for primer design rather than estimating from GC alone.
The formula
count(X) is the number of times base X appears in the sequence after lower-casing and stripping non-base characters. T and U are treated identically — paste DNA or RNA, the result is the same. Ambiguity codes (N, Y, R, W, S, K, M, B, D, H, V) are not counted in either numerator or denominator, so a sequence with many N's is reported on the basis of the unambiguously-called positions only. The result is a percentage in the range 0-100; for short or low-complexity sequences this number is dominated by sampling noise (a 20-mer has a standard error of roughly ±10 percentage points around the underlying GC content), so don't over-interpret three-decimal-place precision on a short input.
Example calculation
- Paste a 32-bp sequence: ATGCATGCATGCATGCGCGCGCATATATATGC.
- Counts: A = 9, T = 7, G = 8, C = 8. Total = 32 valid bases.
- GC = 8 + 8 = 16. GC% = 16 / 32 × 100 = 50.00% — exactly balanced.
Frequently asked questions
Does the calculator handle FASTA format?
Yes. Any line beginning with `>` is treated as a header and excluded from the base count, so you can paste a FASTA record verbatim — the calculator processes only the sequence lines below the header. Multi-record FASTA (several `>` headers in one paste) is concatenated into a single GC % across all sequence lines, which may not be what you want if the records are biologically distinct; in that case run them one at a time. The same parser strips any whitespace (including newlines and tabs), digits (so numbered sequences from GenBank flat files work), and punctuation, so almost any text-format dump of a sequence will give you the right answer.
What does it do with N's and other ambiguity codes?
They're excluded from both the numerator and the denominator, which means the GC % is calculated only over positions where the base is unambiguously called as A, C, G or T/U. So a 100-bp sequence with 80 unambiguous bases (40 GC) and 20 N's reports GC = 50% (40/80), not 40% (40/100). This is the right answer when N's represent "unable to call" — you don't want to bias the GC estimate by counting unknowns as not-GC. If you have a specific reason to want N's in the denominator (e.g. you're comparing against a published number that did include them), strip the N's from your input before pasting and the answer will be the same regardless. Other IUPAC codes (R, Y, W, S, K, M, B, D, H, V) are handled the same way as N — they're ambiguous, so they're skipped.
What's a "good" GC content for a primer?
For standard PCR primers, aim for 40-60% GC. Below 40%, primers tend to bind weakly and you risk poor amplification, especially at standard annealing temperatures. Above 60%, primers can form stable secondary structures (hairpins, primer-dimers) and tolerate single-base mismatches more readily, which can cause off-target amplification. Within that 40-60% window, prioritise other primer-design metrics over fine-tuning the GC: avoid runs of more than three identical bases, ensure GC content is roughly evenly distributed across the primer rather than clustered, and aim for a 3' end with one or two G/C bases (a "GC clamp") to anchor the binding. For primers in genomes with extreme overall GC content (very low-GC bacteria, or GC-rich Streptomyces), strict 40-60% may not be achievable in your target region — in that case match the primer GC to the local genomic GC and rely on stricter Tm matching and higher-temperature annealing to maintain specificity.
How does GC content affect melting temperature?
Higher GC means a higher melting temperature, because GC base pairs hold three hydrogen bonds while AT pairs hold only two. The relationship is real but not directly proportional — it depends strongly on length, salt concentration and the specific neighbouring bases. The classic Wallace rule for short oligos (≤14 bp) is Tm ≈ 4 × (G + C) + 2 × (A + T) °C, which gives a quick mental estimate; for longer oligos a basic formula like Tm ≈ 64.9 + 41 × (GC% − 16.4) / length performs better. Both of these are approximations of the more accurate "nearest-neighbour" method, which uses thermodynamic parameters for each adjacent base-pair stack. For real primer-design work, use a dedicated Tm calculator with the nearest-neighbour algorithm — you'll get values that are reliable to within about 1-2 °C, where the approximations can be 3-5 °C off in extreme cases. As a rough sense-check: a 20-mer at 40% GC melts around 56 °C, the same 20-mer at 60% GC melts around 64 °C, both at standard PCR salt conditions.