MLPA, digitalMLPA — Multiplex Ligation-dependent Probe Amplification

MLPA quantifies up to 50 genomic targets in one reaction. Each target is interrogated by a probe made of two half-oligos that hybridise to immediately adjacent sites; a thermostable ligase joins them only when the ligation junction is a perfect match. A single universal primer pair then amplifies every ligated probe, and because each probe carries a length-tuning stuffer, the products separate by size on capillary electrophoresis - peak area is proportional to target copy number.

Scaling up to NGS — digitalMLPA reads the ligated probes on an Illumina sequencer, so each probe is identified by its sequence rather than its size - pushing a single reaction to 1000 targets, with a per-sample barcode oligo for pooled, multiplexed runs.

The target-specific design is identical for both formats: the LHS/RHS hybridising arms and the ligation junction this tool selects are directly reusable. For a digitalMLPA layout, swap the capillary universal-primer tags (editable below) for the Illumina adapter sequences (P5/P7 + read primer) and add a per-sample barcode oligo; the length-staggering stuffers are then optional, since probes are told apart by sequence, not amplicon size.