Multiplex Ligation-Dependent Probe Amplification (MLPA)

Multiplex ligation-dependent probe amplification (MLPA) is a PCR-based method that detects copy number changes (deletions and duplications) at up to ~50 specific genomic loci in a single reaction. It is widely used for targeted assessment of exon-level deletions and duplications in known disease genes, complementing sequencing approaches that may miss these changes.

• Probe design: Each MLPA probe consists of two half-probes that hybridize to adjacent sequences on the target DNA. Only when both halves bind correctly can they be ligated together by a thermostable ligase, forming a complete probe.
• Amplification: All ligated probes are amplified in a single PCR reaction using a universal primer pair. Each probe produces a fragment of a unique length due to variable-length stuffer sequences.
• Quantification: Fragment analysis by capillary electrophoresis measures the peak height/area for each probe. The relative peak heights are compared to a normal reference sample. A ratio of ~0.5 indicates a heterozygous deletion; a ratio of ~1.5 indicates a heterozygous duplication.
• Multiplex capacity: A single MLPA reaction can interrogate up to ~50 different genomic targets, each represented by a probe of a different size.
• Exon-level resolution: MLPA probes are typically designed for each exon of a gene, enabling detection of single-exon and multi-exon deletions/duplications.
• Commercial kits available: MRC Holland manufactures standardized MLPA probe mixes for hundreds of genes and genomic regions (e.g., P003 for MLH1/MSH2, P245 for microdeletion syndromes, P036/P070 for subtelomeric regions).

• Complement to sequencing: Sanger and NGS may miss heterozygous whole-exon deletions/duplications because the remaining normal allele still generates a sequence. MLPA fills this gap.
• Hereditary cancer syndromes: Detection of large deletions/duplications in BRCA1, BRCA2, MLH1, MSH2, PMS2 (accounts for ~5–10% of pathogenic variants in some of these genes)
• Neuromuscular disorders: DMD (Duchenne/Becker muscular dystrophy): exon-level deletions/duplications account for ~70% of pathogenic variants
• Microdeletion syndromes: Targeted confirmation of specific microdeletion syndromes (e.g., 22q11.2 deletion, Williams syndrome, Smith-Magenis syndrome)
• Methylation-specific MLPA (MS-MLPA): A variant that simultaneously assesses copy number AND methylation status. Critical for imprinting disorders like Prader-Willi syndrome, Angelman syndrome, and Beckwith-Wiedemann syndrome.

• Targeted, not genome-wide: Only detects changes at the specific loci where probes are designed. Unlike CMA, it does not provide genome-wide CNV screening.
• Does not detect balanced rearrangements: Inversions and balanced translocations produce no copy number change and will not be detected.
• Does not detect point mutations: MLPA measures copy number, not sequence changes. However, a point mutation at a probe binding site can cause a false-positive "deletion" due to probe hybridization failure.
• Sequence variants under probes: SNPs or variants at the probe ligation site can interfere with ligation and mimic a deletion (false positive). Confirmatory testing (e.g., a second MLPA kit with different probe placement, or CMA) is recommended.
• Does not determine mechanism: Cannot distinguish tandem duplication from insertional duplication or identify breakpoints precisely.
• Requires high-quality DNA: Degraded DNA can give unreliable results.