Although commonly represented as joining two pairs of ends at once, as in the ligation of restriction enzyme fragments, ligase can also join the ends on only one of the two strands (for example, when the other strand is already continuous or lacks a terminal phosphate necessary for ligation).
Sequencing by ligation relies upon the sensitivity of DNA ligase for base-pairing mismatches.
A mixed pool of probe oligonucleotides is then brought in (eight or nine bases long), labeled (typically with fluorescent dyes) according to the position that will be sequenced.
Based on the fluorescence produced by the molecule, one can infer the identity of the nucleotide at this position in the unknown sequence.
[2][3] Sequencing by ligation can proceed in either direction (either 5'-3' or 3'-5') depending on which end of the probe oligonucleotides are blocked by the label.