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An oligonucleotide is a short nucleic acid chain: an oligomer made from nucleotides, rather than from free nucleobases alone. It may be DNA or RNA. The description concerns chain length and composition, not a guarantee that the chain is single-stranded, naturally occurring or translated into a protein. IUPAC's terminology explicitly connects the word with oligo and nucleotides.^[ref]

For an unmodified linear DNA example, $5^{\prime }\text{-}\mathrm{AGTCG}\text{-}{3}^{\prime }$ has five nucleotides and four connecting phosphodiester bonds. A fully complementary strand is $3^{\prime }\text{-}\mathrm{TCAGC}\text{-}{5}^{\prime }$. Writing that partner in the conventional $5^{\prime }$-to-$3^{\prime }$ direction gives $5^{\prime }\text{-}\mathrm{CGACT}\text{-}{3}^{\prime }$, the reverse complement. Reversing the letters without complementing them would answer a different question. The five-position count also allows $4^5=1024$ possible strings over the standard four-letter DNA alphabet.

Chemical synthesis makes this sequence-level control experimentally useful. In 1994, researchers prepared three-nucleotide building blocks representing codons for all 20 amino acids and incorporated them during automated DNA synthesis. Adding whole triplets allowed mixed oligonucleotides to vary at the codon level rather than only one base at a time.^[ref] This separates designing an individual chain from designing the distribution of sequences in a collection.

Ribosomal RNA oligonucleotide cataloguing compares organisms through collections of short sequences obtained from ribosomal RNA. Instead of determining one complete modern sequence, investigators generated fragments, identified their sequences and compared which longer fragments were shared. The resulting catalog supplied molecular evidence about relationships beyond external appearance.

The 1977 Carl R. Woese and George E. Fox paper describes digestion of small-subunit ribosomal RNA with ribonuclease T1, followed by separation of the products using two-dimensional electrophoresis. Sequencing the resulting oligonucleotides produced a catalog characteristic of the examined material. Comparing catalogs helped establish the distinct position of the methanogenic organisms discussed in the paper. This was an RNA-fragment experiment, not a modern amplified-marker survey.^[ref]

The RNA oligonucleotide association coefficient used nucleotide counts in cataloged sequences of six or more residues:Here $N_A$ and $N_B$ count nucleotides represented by the eligible fragments in the respective catalogs, while $N_{AB}$ counts those represented in their shared portion. It is therefore not simply the fraction of fragment labels found in common, nor the percentage identity of a complete aligned RNA.^[ref]

For an original accounting example, let $N_A=40$, $N_B=60$ and $N_{AB}=25$. Then $S_{AB}=50/100=0.50$. A catalog with fewer recorded nucleotide residues can share the same amount of material while giving a different normalized comparison. Agreement in short fragments alone also need not imply identical full sequences.

The scientific advance was a reproducible molecular comparison usable across morphologically different microbes. Its interpretation still depended on the marker, sampling and method. A catalog similarity supplies evidence for analysis in phylogenetics, not a complete reconstruction of every gene, a direct date of divergence or a guarantee that two organisms have the same physiology.