Introduction And Definition

Multiplex ligation-dependent probe amplification (MLPA) is an advanced, high-throughput, semi-quantitative molecular cytogenetic technique.
It is designed to detect abnormal gene copy numbers, including submicroscopic deletions and duplications.
It can also identify specific point mutations across multiple genomic targets simultaneously.
Unlike standard polymerase chain reaction (PCR) which amplifies the target patient DNA directly, MLPA amplifies specifically designed, hybridized probes.

Core Principles

The method relies on the hybridization of two adjacent target-specific probes to a single DNA strand.
Both probes must be perfectly hybridized to directly adjacent target sequences to undergo enzymatic ligation.
PCR amplification occurs exclusively for these ligated probes.
The amount of amplified probe product is directly proportional to the amount of target DNA present in the patient's sample.

This section contains a target-specific sequence.
It includes a variable-length "stuffer" sequence.
It ends with a universal reverse primer sequence.
The stuffer sequence is crucial for multiplexing.
It ensures that each ligated probe pair has a unique total length, allowing separation by capillary electrophoresis.

The genomic DNA of the patient is heated to 98°C.
This separates the double-stranded DNA into single strands to allow access for the probes.

The specific MLPA probe mix is added to the sample.
The left and right probe oligonucleotides hybridize to their specific adjacent target sequences.
This occurs during an overnight incubation period at 60°C.

A highly specific heat-stable DNA ligase, known as Ligase-65, is introduced.
It covalently links the adjacent left and right probes.
Ligation strictly requires perfect complementarity at the probe junction.
A single nucleotide mismatch at the ligation site prevents successful ligation.

PCR amplification is performed using a single pair of universal fluorescently labeled primers.
Because all probes utilize the same universal primers, amplification bias is minimized.
Unligated probes are not amplified exponentially because they only possess one primer binding site.
The amplified PCR products, or amplicons, are separated based on their unique sizes using capillary electrophoresis.

Raw data peak areas are analyzed using specialized software.
Intra-sample normalization compares each peak against reference control peaks within the same patient sample.
Inter-sample normalization compares the normalized probe signals to those obtained from healthy reference DNA.
The results are reported as a Dosage Quotient (DQ).
A normal copy number of two alleles yields a DQ of approximately 1.0.
A heterozygous deletion yields a DQ of roughly 0.5.
A heterozygous duplication yields a DQ of around 1.5.
A homozygous deletion yields a DQ of 0.

Disease Category	Specific Conditions	Target Gene Or Region
Neuromuscular Disorders	Spinal muscular atrophy (SMA)	SMN1 and SMN2 copy number quantification.
	Duchenne and Becker muscular dystrophy	DMD exonic deletions and duplications.
Neurodevelopmental Disorders	Rett syndrome	MECP2 large deletions or duplications.
Peripheral Neuropathies	Charcot-Marie-Tooth disease type 1A and Hereditary neuropathy with liability to pressure palsies	PMP22 gene duplication or deletion.
Imprinting Disorders	Prader-Willi syndrome, Angelman syndrome, Beckwith-Wiedemann syndrome	Evaluated using Methylation-Specific MLPA (MS-MLPA) to assess methylation patterns and copy number.
Microdeletion Syndromes	DiGeorge syndrome, Williams syndrome	Targeted screening for classical contiguous gene deletion syndromes.

Feature	Description
Advantages	High multiplexing capacity allows analysis of up to 50 to 60 distinct genomic targets in a single reaction.
	It is highly cost-effective and provides a faster turnaround time compared to next-generation sequencing for targeted testing.
	It offers a high resolution capable of detecting single exon deletions or duplications.
	It requires very low genomic DNA input, functioning with as little as 20 nanograms.
Limitations	It utilizes a targeted approach, meaning it only detects copy number changes in the specific sequences targeted by the probes.
	It cannot detect balanced chromosomal translocations or inversions.
	It is susceptible to benign single nucleotide polymorphisms at the probe ligation site, which can falsely indicate a deletion known as allele drop-out.
	It does not broadly detect point mutations, intronic variants, or triplet repeat expansions.