Biology calculators

GC Content Calculator

Updated Oct 8, 2026 By Infinity Calculator
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Sequence Input

Accepts a raw sequence, a single FASTA entry, or multi-FASTA. IUPAC degenerate codes (R, Y, S, W, K, M, B, D, H, V, N) are supported. Input is read case-insensitively; minimum 10 and maximum 100,000 nucleotides per sequence.
Valid nucleotides: 0 Quick GC preview: 0.00% Sequences detected: 1 Running preview only — press Calculate for the full analysis.

Analysis Options

Sequence type
Auto-detect uses the presence of U vs. T.
Any IUPAC letters, e.g. GC, AT, AU, GCU. T and U are treated as equivalent.
Minimum 10; must be smaller than the sequence length.
All Tm outputs are converted to this unit.
Silently strips spaces, line breaks, digits and gap characters (- . * _ :) before analysis.
When unchecked, GC% uses only A, C, G, T/U counts as the denominator.
Primary Result
Per-Base Breakdown
Step-by-Step Solution
GC Content Distribution (Sliding Window)
Base Composition
Analyze a Sub-Region
Sequence Viewer
CpG Island Analysis

Introduction

DNA is made of four bases: A, T, G, and C.1 GC content is the percent of bases in a sequence that are G (guanine) or C (cytosine). This free GC content calculator counts those bases and shows the answer as a percent.

GC content matters because G and C pair with three hydrogen bonds, while A and T pair with only two.2 So DNA with more G and C holds together more tightly and needs more heat to split apart.3 That is why scientists check GC content when they design PCR primers, pick annealing temperatures, or compare genes between species.

Paste a raw DNA or RNA sequence, or a FASTA file with many sequences. The tool then gives you:

  • GC% and AT% for each sequence
  • A count and percent for every base, including IUPAC codes like N, R, and Y
  • Melting temperature (Tm) in °C, °F, or K
  • A sliding window chart that shows where GC is high or low
  • CpG island detection and a color-coded sequence viewer

You can also pick your own bases to count, check just one part of the sequence, and download the results as a CSV file. Step-by-step math is shown, so you can see how each number was found.

How to use our GC Content Calculator

Paste a DNA or RNA sequence, pick a few options, and press Calculate. The tool shows your GC content percent, AT content, base counts, melting temperature (Tm), a sliding window GC chart, and any CpG islands it finds.

DNA / RNA sequence or FASTA input: Type or paste your sequence in the big box. You can use a plain sequence, one FASTA entry, or many FASTA entries at once. IUPAC codes like N, R, and Y are fine. Each sequence needs at least 10 bases and no more than 100,000.

Sequence type: Choose Auto, DNA, or RNA. Auto looks for U or T and picks for you. Pick DNA or RNA yourself if you already know.

Calculate content for bases: Enter the letters you want counted. Leave it as GC for normal GC content, or try AT, AU, or any other IUPAC letters. T and U count as the same base.

Sliding window size (bp): Set how many bases each window covers on the GC chart. Use 10 or more, and keep it smaller than your sequence. Small windows show fine detail; big windows show smooth trends.

Melting temperature unit: Pick Celsius, Fahrenheit, or Kelvin. Every Tm result switches to that unit.

Auto-clean input: Keep this checked to drop spaces, line breaks, numbers, and gap marks like - . * _ before the math runs.

Include degenerate bases in the denominator: Check this to count codes like N and R in the total. Leave it off to use only A, C, G, and T/U as the total.

Sub-region start and end: After you calculate, type a start and end position and click Analyze region to get GC content for just that part of the sequence. It also gets marked in the sequence viewer.

What Is GC Content?

DNA is made of four bases: adenine (A), thymine (T), guanine (G), and cytosine (C).1 In RNA, uracil (U) takes the place of T.1 GC content is the percent of bases in a sequence that are either G or C. It is one of the most basic facts you can learn about a piece of DNA or RNA.

The GC Content Formula

GC content is simple math:

GC% = (G + C) ÷ (A + T + G + C) × 100

So a 100-base sequence with 30 G's and 25 C's has a GC content of 55%. The leftover part is the AT content, which is 45%.

Why GC Content Matters

G and C pair with three hydrogen bonds. A and T pair with only two.2 More G and C pairs means the DNA strands hold together more tightly. This changes how the DNA acts in the lab and in the cell:

  • Melting temperature (Tm): High GC DNA needs more heat to split into two strands. This matters a lot for PCR primers.
  • Primer design: Most good primers land near 40% to 60% GC.4 Very low GC primers bind weakly. Very high GC primers can stick to the wrong spots or form hairpins.3
  • Gene finding: Most gene promoters in vertebrates sit in CpG islands, which are richer in G and C than the DNA around them.12
  • Species clues: Different organisms have very different average GC. Bacterial genomes range from less than 20% to more than 70% GC.9 Human DNA averages about 41%.8

Melting Temperature Rules

Two common quick formulas are used here:

  • Wallace rule (best for short primers, 14 bases or fewer): Tm = 2(A + T) + 4(G + C).5 Some suppliers subtract 7 °C from this result for primers used in solution, as in PCR.5
  • Basic formula (better for longer sequences): Tm = 64.9 + 41 × (G + C − 16.4) ÷ length.6

Both are estimates. They assume set conditions of 50 nM primer, 50 mM sodium and pH 7.0.6 They also count bases only, so they ignore mismatches. Treat them as a starting point, not a final answer.

CpG Islands

A CpG site is a C followed by a G on the same strand. In human DNA they are rare, at about one-fifth of the number you would expect from the amounts of C and G.8 But short stretches called CpG islands are packed with them, and they often sit at the start of genes.11 The standard rule (Gardiner-Garden and Frommer) calls a region a CpG island when it is at least 200 bases long, has at least 50% GC, and has a CpG observed/expected ratio above 0.6.10 CpG islands are important because methylation at these sites can switch genes off.12

Degenerate (IUPAC) Bases

Sometimes a sequence uses letters other than A, C, G, T, and U. These IUPAC codes stand for more than one possible base, such as R (A or G), Y (C or T), S (G or C), W (A or T), and N (any base).7 They show up in consensus sequences and in reads where the base was unclear. You can choose to count them in the total or leave them out.

Reading Your Results

GC ContentWhat It Suggests
Below 35%AT-rich. Common in intergenic DNA, some bacteria, and AT-rich genomes like Plasmodium.
35% to 60%Balanced. Typical for most genes and good for primer design.
Above 60%GC-rich. Often promoters, CpG islands, or GC-rich organisms. Can be hard to amplify by PCR.3

Sliding Windows

One average number hides a lot. A sliding window moves a short frame (say 100 bases) along the sequence and measures GC in each step. This shows where GC rises and falls, which helps you spot promoters, CpG islands, repeats, and joins between different pieces of DNA.

Formulas used

Target base content (e.g. GC%)
\text{GC\%} = \frac{n_{G} + n_{C}}{n_{\text{denom}}} \times 100
AT content
\text{AT\%} = \frac{n_{A} + n_{T/U}}{n_{\text{denom}}} \times 100
Denominator (with or without degenerate bases)
n_{\text{denom}} = n_{A} + n_{C} + n_{G} + n_{T/U} \; (+\; n_{\text{degenerate}})
GC/AT ratio
R_{GC/AT} = \frac{n_{G} + n_{C}}{n_{A} + n_{T/U}}
Melting temperature — Wallace rule (length \le 14) 5
T_m = 2\,(n_{A} + n_{T/U}) + 4\,(n_{G} + n_{C})
Melting temperature — basic formula (length > 14) 6
T_m = 64.9 + 41 \times \frac{(n_{G} + n_{C}) - 16.4}{n_{A} + n_{T/U} + n_{G} + n_{C}}
CpG observed/expected ratio 18
\frac{O}{E} = \frac{n_{CpG} \times L}{n_{C} \times n_{G}}
Temperature unit conversion 16
T_{^{\circ}F} = T_{^{\circ}C} \times \frac{9}{5} + 32, \qquad T_{K} = T_{^{\circ}C} + 273.15

Frequently asked questions

What is a good GC content for PCR primers?

Most primers work best at 40% to 60% GC.4 Aim for a melting temperature (Tm) of 50 to 60 °C as well.4

  • Under 40% GC: the primer binds weakly and may need a lower annealing temperature.
  • Over 60% GC: the primer can stick to the wrong places or fold on itself.

Also try to keep the forward and reverse primer Tm values within about 5 °C of each other.4

What is a GC clamp in primer design?

A GC clamp is one or two G or C bases at the 3' end of a primer.4 Because G-C pairs have three hydrogen bonds, they grip the template tightly at the exact spot where the polymerase starts building.3

A common rule of thumb is one or two G/C bases among the last five positions, and no more than three. A 3' end with too many G/C bases binds too tightly and can cause false priming.

Why is GC-rich DNA hard to amplify by PCR?

GC-rich DNA holds together tightly and can form hairpins and other folds.3 The strands may not fully separate during denaturation, so the primers cannot reach the template.3

Common fixes:

  • Add DMSO, glycerol or betaine, which reduce the folds that can stall the polymerase.3
  • Try a higher annealing temperature, which can also help pull folds apart.3
  • Use a polymerase or buffer made for GC-rich targets.3

What is the average GC content of the human genome?

The human genome averages about 41% GC.8 But the value is not the same everywhere. Large regions called isochores have been sorted into five classes, from under 38% to over 52% GC.8

Chromosome 19 is the most GC-rich human chromosome at around 48%, while chromosome 4 sits near 38%. Gene-packed regions usually have higher GC than gene-poor regions.

Why do bacteria have such different GC content?

Bacterial GC content ranges from less than 20% to more than 70%.9

The spread has usually been put down to differences in mutation, but a study of 149 bacterial species found signs that natural selection pushes GC content up as well.9 For example, Escherichia coli is near 50%, Mycoplasma near 25%, and Streptomyces above 70%.

Does high GC content mean an organism lives in a hot place?

Not for whole genomes. Many heat-loving microbes have low genomic GC, so total GC does not predict growth temperature.13

The link is real for structural RNA, though. The GC content of structural RNAs, such as ribosomal and transfer RNA, rises with a microbe's best growth temperature.13

What is GC skew and how is it different from GC content?

GC content counts G and C together. GC skew compares them: (G − C) ÷ (G + C) on one strand.14

In many bacterial genomes the skew flips sign at the origin of replication and again at the terminus.14 Because of that, biologists use GC skew plots to find where DNA copying starts, something plain GC content cannot show.14

Why do coding regions often have higher GC content?

Two main reasons:

  • Codon choice: the third base of a codon can often change without changing the protein. Many species favor G or C there.
  • Repair bias: when DNA is exchanged during recombination, the repair step tends to favor G/C over A/T, which raises GC content in regions that recombine often.17

This is why a jump in GC on a sliding window plot can point to a gene or a promoter.

Does GC content affect DNA sequencing results?

Yes. Regions that are very GC-rich or very AT-rich usually get fewer sequencing reads.15 This is called coverage bias, and it mostly comes from the PCR step used to build sequencing libraries.15

Low coverage means variants in those regions are easier to miss. Better PCR conditions during library preparation cut the bias sharply.15

How do I find AT content if I know the GC content?

Subtract from 100. AT% = 100 − GC%

So a sequence with 58% GC has 42% AT. This works because A, T, G, and C are the only four bases counted. If the sequence has unclear bases like N, the two numbers may not add to exactly 100 unless those bases are left out of the total.

Why are CpG islands found at the start of so many genes?

About 70% of annotated gene promoters are linked to a CpG island.12 When the C bases in an island stay unmethylated, the DNA stays open and the gene can be switched on.

When those C bases get methylated, the gene is usually turned off.12 Tumor cells often methylate CpG islands in front of protective genes, which is why these regions are studied so closely in cancer research.12

How does GC content change DNA melting temperature?

Each extra percent of GC raises the melting temperature by roughly 0.4 °C for long DNA. A G-C pair has three hydrogen bonds, and stacking between neighboring G/C bases is also stronger, so more heat is needed to pull the strands apart.

Salt matters too. Raising salt shields the negative backbone and pushes Tm higher, so always note the buffer when you compare Tm values.

Sources

  1. Clark MA, Douglas M, Choi J. Biology 2e, 3.5 Nucleic Acids. OpenStax. 2018;section 3.5, Table 3.2. Accessed September 29, 2026.
  2. Clark MA, Douglas M, Choi J. Biology 2e, 14.2 DNA Structure and Sequencing. OpenStax. 2018;section 14.2. Accessed September 29, 2026.
  3. Gibson J. Four tips for PCR amplification of GC-rich sequences. New England Biolabs. 2022. Accessed September 29, 2026.
  4. Protocol - How to Design Primers. Addgene. Accessed September 29, 2026.
  5. Oligonucleotide Melting Temperature. Sigma-Aldrich. Basic method. Accessed September 29, 2026.
  6. What formula is used to calculate melting temperature (Tm)? Rosalind, Inc.. Accessed September 29, 2026.
  7. BLAST topics: FASTA format. National Center for Biotechnology Information. nucleic acid codes. Accessed September 29, 2026.
  8. International Human Genome Sequencing Consortium. Initial sequencing and analysis of the human genome. Nature. 2001;409:860-921. doi:10.1038/35057062. Accessed September 29, 2026.
  9. Hildebrand F, Meyer A, Eyre-Walker A. Evidence of selection upon genomic GC-content in bacteria. PLoS Genetics. 2010;6(9):e1001107. doi:10.1371/journal.pgen.1001107. Accessed September 29, 2026.
  10. Takai D, Jones PA. Comprehensive analysis of CpG islands in human chromosomes 21 and 22. Proceedings of the National Academy of Sciences. 2002;99(6):3740-3745. doi:10.1073/pnas.052410099. Accessed September 29, 2026.
  11. Gardiner-Garden M, Frommer M. CpG islands in vertebrate genomes. Journal of Molecular Biology. 1987;196(2):261-282. doi:10.1016/0022-2836(87)90689-9. Accessed September 29, 2026.
  12. Deaton AM, Bird A. CpG islands and the regulation of transcription. Genes & Development. 2011;25(10):1010-1022. doi:10.1101/gad.2037511. Accessed September 29, 2026.
  13. Hurst LD, Merchant AR. High guanine-cytosine content is not an adaptation to high temperature: a comparative analysis amongst prokaryotes. Proceedings of the Royal Society B: Biological Sciences. 2001;268(1466):493-497. doi:10.1098/rspb.2000.1397. Accessed September 29, 2026.
  14. Grigoriev A. Analyzing genomes with cumulative skew diagrams. Nucleic Acids Research. 1998;26(10):2286-2290. doi:10.1093/nar/26.10.2286. Accessed September 29, 2026.
  15. Aird D, Ross MG, Chen W, Danielsson M, Fennell T, Russ C, Jaffe DB, Nusbaum C, Gnirke A. Analyzing and minimizing PCR amplification bias in Illumina sequencing libraries. Genome Biology. 2011;12:R18. doi:10.1186/gb-2011-12-2-r18. Accessed September 29, 2026.
  16. SI Units – Temperature. National Institute of Standards and Technology. Accessed September 29, 2026.
  17. Duret L, Galtier N. Biased Gene Conversion and the Evolution of Mammalian Genomic Landscapes. Annual Review of Genomics and Human Genetics. 2009;10(1):285-311. doi:10.1146/annurev-genom-082908-150001. Accessed October 8, 2026.
  18. Lin TY, Chen HY, Cheng YH, Lu CW, Ma HY, Hou HC, et al. Dysregulated methylation of the human BOLL promoter hinders germ cell development from spermatogonia to spermatids. Molecular Biology Reports. 2026;53(1):477. doi:10.1007/s11033-026-11633-0. Accessed October 8, 2026.

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