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Sequence Statistics.

Analyze DNA or RNA sequences: GC content, molecular weight, melting temperature, codon usage, ORF finder, and reverse complement.

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Validated2026-04-05
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When to use

  • Quickly assess GC content for primer design or genome comparisons
  • Calculate molecular weight for oligonucleotide ordering and mass-spec verification
  • Estimate melting temperature for PCR optimization
  • Find open reading frames in a cloned insert or cDNA sequence
  • Analyze codon usage for expression optimization or evolutionary studies

Do not use for

  • For genome-scale analysis (>100 kb) — use dedicated bioinformatics tools like BLAST or Geneious
  • As a substitute for proper gene prediction with promoter and RBS analysis
  • For Tm estimation requiring nearest-neighbor thermodynamics with salt/mismatch corrections

Clean your sequence before analysis

FASTA headers, line numbers, spaces, and non-nucleotide characters will be automatically stripped, but always verify the cleaned sequence length matches your expectation. Ambiguity codes (N, R, Y, etc.) are removed during cleaning.

Wallace rule Tm is only for short oligos

The simple formula Tm = 2(A+T) + 4(G+C) is accurate only for oligonucleotides shorter than ~14 nt. For PCR primers (18–25 nt), use the salt-adjusted formula. For exact Tm with mismatches, use nearest-neighbor thermodynamic tools.

ORFs are candidates, not confirmed genes

Finding an ATG-to-stop reading frame does not prove a gene exists. Short ORFs occur by chance. True gene identification requires experimental evidence or comparative genomics. The tool reports the longest ORF per frame as the most likely candidate.

Codon frequency depends on reading frame

Codon usage is computed in reading frame 0 (starting from the first nucleotide). If your sequence does not start at a codon boundary, trim it first or note that the codon counts reflect frame 0 only.

1

Method

GC content by direct base counting. Molecular weight from nucleotide monophosphate masses with phosphodiester bond water loss correction. Melting temperature via the Wallace rule (<14 nt) or salt-adjusted basic formula (14–100 nt). Codon usage from the standard genetic code (NCBI translation table 1). ORF finder scans all three forward reading frames for the longest ATG-to-stop sequences.

2

Validated

Last validated 2026-04-05. Calculations are designed for planning and documentation support; verify procurement decisions against manufacturer specifications or institutional SOPs.

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How to cite

How to Cite

ConductScience DNA/RNA Sequence Statistics Calculator (v1.0). ConductScience, Inc. 2026. Available at: https://conductscience.com/tools/sequence-stats-calculator

Breslauer KJ, et al. Predicting DNA duplex stability from the base sequence. Proc Natl Acad Sci USA. 1986;83(11):3746–3750.

Sharp PM, Li WH. The codon adaptation index — a measure of directional synonymous codon usage bias. Nucleic Acids Res. 1987;15(3):1281–1295.

Sequence Analysis Fundamentals

DNA and RNA sequence analysis begins with basic composition metrics that reveal structural and functional properties:

GC Content — The ratio of guanine + cytosine to total bases, expressed as a percentage. G-C base pairs form three hydrogen bonds (vs. two for A-T/A-U), so higher GC content increases thermal stability. GC content varies by genome region: coding sequences, CpG islands, and rRNA genes tend to be GC-rich.
Molecular Weight — Calculated from the sum of individual nucleotide monophosphate masses minus water released during polymerization. Essential for stoichiometric calculations, gel electrophoresis estimation, and mass spectrometry verification.
Melting Temperature (Tm) — The temperature at which half of the DNA duplexes denature. Critical for PCR primer design (annealing temperature ≈\approx Tm − 5°C), hybridization stringency, and understanding in vivo stability.

Codon Usage & Open Reading Frames

Beyond composition, the coding potential of a sequence is assessed through codon analysis and ORF identification:

Codon Usage — The genetic code is degenerate: 61 sense codons encode 20 amino acids. Organisms exhibit codon usage bias, preferring certain synonymous codons. High-expression genes tend to use codons matching abundant tRNAs. The Codon Adaptation Index (CAI) quantifies how well a gene's codon usage matches the host organism.
Open Reading Frames (ORFs) — An ORF is a continuous stretch of codons beginning with ATG (start) and ending with a stop codon (TAA, TAG, or TGA). Three forward reading frames exist for each strand (six total for double-stranded DNA). The longest ORFs in each frame are candidates for protein-coding genes, though true gene identification requires promoter analysis, ribosome binding sites, and comparative evidence.
Reverse Complement — DNA is antiparallel: the two strands run 5'→3' and 3'→5'. The reverse complement converts between strands while maintaining the 5'→3' convention, essential for primer design and annotation.

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