Multiplex ligation-dependent probe amplification (MLPA)
Multiplex ligation-dependent probe amplification (MLPA) uses pairs of probes to detect specific sections of the genome. It is mainly used to identify exon-level copy number variation.
What is MLPA?
MLPA is commonly used to diagnose conditions that are predominantly caused by exon-level copy number variation (deletions and duplications), for example spinal muscular atrophy (SMA).
Alternatively, it can be used in combination with next-generation sequencing to examine genes that may contain single nucleotide variants or copy number variants (for example, BRCA1, BRCA2 in breast cancer).
It may also be used:
- to test family members for known familial deletions/duplications;
- to refine the breakpoints of intragenic copy number variants detected by microarrays;
- occasionally, to test for specific single nucleotide variants (for which probes are included in the kit); and
- in methylation-specific MLPA (MS-MLPA), to determine the methylation status of imprinted or promoter regions at which aberrant methylation is associated with disease. For clinical examples, see this Knowledge Hub article.
How does MLPA work?
- MLPA uses pairs of oligonucleotide probes – small molecules of DNA that are designed to hybridise with a specific genomic sequence. Multiple pairs of probes can be multiplexed in the same reaction, for example to screen for all exons of a gene.
- The probes bind only where their exact target sequence is present. If the probes in a pair bind immediately next to each other, their ends ligate (join together).
- There is one primer binding site on the end of each of the probes in the pair. PCR amplification therefore only occurs if probes in a pair have ligated.
- Amplification products are separated by length using capillary electrophoresis.
- The amount of amplification product in MLPA is proportional to the amount of target sequence present in the sample. Differences in peak heights of amplification products of sample target probes can be compared with those from a reference sample with a known copy number to detect dosage abnormalities in the sample (e.g. exon deletions/duplications).
Methylation-specific MLPA (MS-MLPA)
- MS-MLPA can be used to determine the methylation status of an imprinted or promoter region.
- Patient DNA is used in two reactions. One reaction is a standard MLPA reaction (as above). For the second reaction, prior to the MLPA, a methylation-sensitive endonuclease (restriction digest enzyme) is applied.
- Methylated regions of DNA are protected from digestion and amplification products result; unmethylated regions are digested (broken down) and therefore no amplification product is formed.
- MLPA peak heights are compared between the reaction that has been exposed to the methylation-sensitive endonuclease and the reaction that has not. Peaks that occur in both show where methylation occurs, those that occur in only the undigested reaction are not methylated.
- Both CNVs and methylation status can be determined.
For a more detailed look at how MLPA works, see ‘Resources for clinicians’ below.
Advantages and limitations of MLPA
- Gold standard for exon-level CNV detection (alongside some microarrays)
- Robust; fast; cost-effective.
- High resolution, accurate detection of single exon deletions/duplications.
- Can be designed to include specific single nucleotide variants (SNVs).
- Can be modified to detect methylation (MS-MLPA).
- Higher throughput and less laborious than FISH.
- Commercial MLPA kits are not available for all genes.
- Limited number of target regions per kit (digital MLPA may be able to overcome this).
- Where a duplication is detected, the location or orientation of the duplicated material is not known.
- Will not detect balanced rearrangements.
- May not detect mosaicism.
- May miss small/intronic deletions that are located outside of the regions targeted by the probes in the kit.
- If the deletion/duplication extends beyond the region targeted by the probes in the kit, a secondary method may be required to investigate this (for example, microarrays).
- Does not detect SNVs (unless specifically targeted by MLPA probes).
- MRC Holland: The principles of MLPA explained
- Stuppia L, Antonucci I, Palka G and others. ‘Use of the MLPA assay in the Molecular Diagnosis of Gene Copy Number Alterations in Human Genetic Diseases’. International Journal of Molecular Sciences 2012: volume 13, issue 3, pages 3,245-76. doi: 10.3390/ijms13033245