ORF

An open reading frame (ORF) is a continuous stretch of nucleotides that begins with a start codon and ends with a stop codon, with no in-frame stop codon in between. In DNA, the start codon is typically ATG and the stop codons are TAA, TAG, or TGA; in RNA, these correspond to AUG, UAA, UAG, and UGA. An ORF can be read in any of the three reading frames on either strand of the double helix, yielding six possible frames in total. An ORF is a candidate for encoding a protein, though its existence alone does not prove that a functional protein is produced.

ORFs are central to genome annotation and protein-coding gene prediction. In prokaryotes, many ORFs directly correspond to genes because their genomes are typically compact and introns are absent. In eukaryotes, genes are often interrupted by introns, and many genomic ORFs may not be translated into proteins. Consequently, a large number of ORFs found in a genome may reflect noncoding sequences, random chance, pseudogenes, or regulatory elements such as upstream ORFs.

Identification of ORFs is primarily computational. Methods scan sequences in all reading frames to locate start and stop codons and apply filters such as minimum length. Widely used tools include ORF Finder, Prodigal, GeneMark, and AUGUSTUS. Prediction is frequently integrated with other evidence, including transcript data, homology to known proteins, codon usage patterns, and ribosome profiling, to distinguish true protein-coding regions from incidental ORFs. Small ORFs (sORFs) and upstream ORFs (uORFs) can be functional, adding complexity to annotation efforts and underscoring that an ORF alone is not definitive evidence of protein expression.