GC Content Calculator

Work out the GC content of a DNA or RNA sequence, its base composition and molecular weight, and see how GC rises and falls along the sequence in a sliding window.

Sequence tool

Count the bases

DNA or RNA

Bare letters, a FASTA record or a GenBank record. Numbers and spaces from a listing are ignored.

Window

bases

How many bases each point of the plot averages.

Step

bases

How far the window moves between points.

Paste a DNA or RNA sequence to see its GC content.

The formula

GC% = (G + C) ÷ (A + T + G + C) × 100

GC skew = (G − C) ÷ (G + C)

G, C, A, T: the count of each base; U counts as T in RNA
S, W: IUPAC codes that count as G or C (S) and as A or T (W); other codes are left out of the count
window: the stretch of bases GC is averaged over, moved along by the step

Good to know

GC content runs from under 25% in some Plasmodium genomes to over 70% in Streptomyces. Human DNA averages about 41%, higher in gene-rich regions, and a coding sequence is often higher than the genome around it.
Each G·C pair has three hydrogen bonds and stacks more strongly than A·T, so GC-rich DNA melts higher and a primer's Tm rises with its GC. Primers are usually designed at 40–60% GC.
GC skew changes sign at the origin and the terminus of a bacterial chromosome, because the leading strand is richer in G. On a short sequence it means little.
The molecular weight is for the bases as given, with a 5′ phosphate on each strand, as an average mass. A synthetic oligo without the phosphate weighs 61.96 Da less per strand.

In the dictionary: nucleotide, genome and DNA.

Worked example

Human insulin mRNA (NM_000207.3, 465 nt)

01A 91, C 156, G 141, T 77
02GC = (141 + 156) ÷ 465 = 0.639
0363.9% GC; the coding sequence alone, 60–392, is 64.6%

63.9% GC, well above the human average of about 41%: a GC-rich gene, as insulin is.

How it works

The reasoning behind it

GC content is simply the share of bases that are G or C. It is counted over the bases that say which they are: an N tells nothing and is left out, while S (C or G) counts as GC and W (A or T) as AT.

GC varies along a sequence, so the tool also averages it over a window that slides along in steps and draws the result. A dip marks an AT-rich stretch such as a promoter or an origin; a peak marks a CpG island or a GC-rich coding region.

GC matters in the lab. GC-rich templates need a higher annealing or denaturation temperature, often an additive such as DMSO, and resist sequencing; primers want a moderate GC and a balanced 3′ end. In genomes, GC content tracks codon use, gene density and, in bacteria, the lineage.

Questions

Frequently asked

What is a normal GC content?

It depends on the organism. Human DNA is about 41% GC, E. coli about 51%, Plasmodium falciparum under 20%, Streptomyces over 70%. Within a genome, genes and CpG islands run higher than the sequence between them.

How does GC content affect PCR?

GC-rich templates and primers melt at higher temperatures and form stable secondary structures, so they need a higher annealing temperature, sometimes a higher denaturation temperature, and often DMSO or betaine. AT-rich templates mean low primer Tm values and more mispriming.

What is GC skew?

(G − C) ÷ (G + C) in a window: whether the strand has more G than C. In bacteria the leading strand of replication is G-rich, so the skew flips sign at the origin and the terminus, which is one way they are found.

Does the calculator count N and other codes?

N, R, Y and the other ambiguity codes are left out of both the numerator and the denominator, except S, which counts as GC, and W, which counts as AT. The base counts show every letter, including the ambiguous ones.

Research

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