Oligonucleotides require a 5' phosphate for ligation to occur. This phosphate can be added enzymatically using polynucleotide kinase, or can be added chemically during oligonucleotide synthesis. Phosphorylated oligos are also useful in changing the susceptibility of a sequence to hydrolysis by an exonuclease, that's why FRET probes labeled with an acceptor fluorophore require a 3' phosphate.

Phosphates can be added at the 5' and/or 3' end of an oligo.