ORFs

An open reading frame (ORF) is a stretch of nucleotides that has the potential to be translated into a polypeptide. In DNA, an ORF is defined as a sequence beginning with a start codon and ending with a stop codon, and it must be in-frame, i.e., the triplet codons are read in groups of three without encountering a stop codon. Because DNA is double stranded and has three possible reading frames per strand, six reading frames exist in total. An ORF can exist on either strand and in any frame.

In practice, ORFs are identified during genome annotation by scanning for start and stop codons and evaluating potential coding capacity. In bacteria and organelles, the start codon is commonly AUG, though alternative start codons may be used; in eukaryotes, initiation context (Kozak sequence) influences recognition of the start codon, and prokaryotes may rely on Shine-Dalgarno sequences upstream of the start codon.

Because short ORFs occur by chance, many annotations apply a minimum length threshold (for example 100 codons) to focus on likely protein-coding genes. However, smaller ORFs, termed short or small ORFs (sORFs), have been found to encode functional peptides, and they complicate annotation.

An ORF is not guaranteed to be a gene; it may be nonfunctional, a pseudogene, or a regulatory element. Some genomic regions contain overlapping ORFs or alternate reading frames, reflecting complex transcription and translation. Experimental evidence such as transcript abundance, ribosome profiling, or proteomics supports coding status.

ORF annotation is central to genome annotation, comparative genomics, and gene discovery, including in prokaryotic, eukaryotic, organellar, and viral genomes, as well as metagenomic datasets.