GC Content Calculator
GC content is the fraction of bases in a nucleic acid sequence that are guanine (G) or cytosine (C), expressed as a percentage: GC% = (G + C) / total bases x 100. It is a fundamental property of DNA and RNA sequences that influences the thermal stability of double-stranded nucleic acids, PCR primer design, and codon usage bias analysis. Paste or type any DNA or RNA sequence (IUPAC letters; spaces, numbers, and non-base characters are ignored) and the calculator will count each base and report GC content, AT content, and a simple melting temperature estimate for short primers.
GC content formula
GC% = (G + C) / (A + T + G + C) x 100
Tm (short oligo) = 2(A+T) + 4(G+C) degrees Celsius
The Tm formula (Wallace rule) is an approximation for oligonucleotides under 20 bases in standard salt conditions (50 mM NaCl). For longer sequences, use the nearest-neighbor thermodynamic model.
GC content guidelines
- PCR primers: aim for 40-60% GC content for balanced annealing.
- High GC (above 60%) can cause secondary structure issues and may require DMSO or betaine additives.
- Low GC (below 40%) lowers Tm and can reduce primer specificity.
- The ideal Tm for PCR primers is typically 55-65 degrees Celsius.
- Both DNA (T) and RNA (U) sequences are handled identically; T and U are both counted as AT-pair bases.
Frequently asked questions
What is GC content?
GC content is the percentage of bases in a DNA or RNA sequence that are guanine (G) or cytosine (C). It is calculated as (G + C) / (A + T + G + C) x 100. GC content influences melting temperature, codon usage, genome stability, and primer design.
Why does GC content matter for PCR primers?
GC content affects primer melting temperature (Tm). A GC content of 40-60% is generally recommended for PCR primers. GC pairs form three hydrogen bonds versus two for AT pairs, making them stronger. Higher GC content raises Tm. Very high GC (above 70%) can cause secondary structures; very low (below 30%) lowers Tm.
How does GC content relate to melting temperature?
A simple approximation for short oligonucleotides (less than 20 bases) is Tm = 2(A+T) + 4(G+C) degrees Celsius. For longer sequences, the Wallace rule or nearest-neighbor thermodynamics provides more accurate estimates.
What is a typical genome GC content?
GC content varies widely by organism. Human genome: approximately 41%. E. coli: approximately 51%. Mycobacterium tuberculosis: approximately 65% (high GC organism). Plasmodium falciparum: approximately 20% (low GC organism). Knowing genome GC helps interpret codon usage bias.
Does GC content calculation treat RNA differently from DNA?
The calculation is the same: (G + C) / total bases x 100. In RNA, uracil (U) replaces thymine (T) and both are counted as AT-pair equivalents. This calculator accepts both DNA (A, T, G, C) and RNA (A, U, G, C) sequences.
Official sources
- NCBI/NIH: NCBI Primer-BLAST tool and documentation.
- NCBI Bookshelf: Molecular biology of the gene reference.
Reviewed by the CalculatorHub team, edited by James Graham, 15 June 2026. See our methodology.