High-throughput, semi-quantitative molecular technique designed to detect abnormal gene copy numbers (deletions and duplications) and specific point mutations across multiple genomic targets simultaneously.
Rapid diagnostic test capable of delivering precise clinical results within 1 to 2 days.
Amplifies specifically designed, hybridized probes rather than amplifying the target patient DNA directly.
Core Principles And Probe Design
Relies on the hybridization of two adjacent target-specific probes to a single DNA strand.
Enzymatic ligation occurs only when both probes are perfectly hybridized to directly adjacent target sequences.
PCR amplification occurs exclusively for the ligated probes, generating an amount of amplified product directly proportional to the target DNA present in the sample.
Structural Components Of MLPA Probes
Probe Element
Characteristics
Left Probe Oligonucleotide (LPO)
Contains a universal forward primer sequence and a target-specific sequence.
Right Probe Oligonucleotide (RPO)
Contains a target-specific sequence, a variable-length stuffer sequence, and a universal reverse primer sequence.
Stuffer Sequence
Determines the unique total length of the final amplified fragment, allowing multiplexing and size-based separation via capillary electrophoresis.
Sequential Methodology
DNA Denaturation: Patient genomic DNA (50-250 ng) is heated to 98°C to separate double-stranded DNA into single strands.
Hybridization: The probe mix is added, allowing left and right probe oligonucleotides to hybridize to their specific adjacent target sequences during an overnight incubation at 60°C.
Ligation: A heat-stable DNA ligase (Ligase-65) covalently links the adjacent probes. Strict perfect complementarity is required; a single nucleotide mismatch prevents ligation.
Amplification: PCR is performed using a single pair of universal fluorescently labeled primers. Because all probes share universal primers, amplification bias is minimized. Unligated probes possess only one primer binding site and do not amplify exponentially.
Fragment Separation: Amplified PCR products are separated by size using capillary electrophoresis, generating a sequential pattern of peaks known as an electropherogram.
Data Analysis And Normalization
Raw peak areas or heights undergo intra-sample normalization against internal control probes and inter-sample normalization against healthy reference DNA.
Dosage Quotient (DQ)
Copy Number Interpretation
~1.0
Normal copy number (2 alleles).
~0.5
Heterozygous deletion (1 allele).
~1.5
Heterozygous duplication (3 alleles).
0
Homozygous deletion (0 alleles).
Clinical Applications In Pediatrics
Neuromuscular And Neurodevelopmental Disorders
Spinal Muscular Atrophy (SMA): Gold standard first-line diagnostic test. Accurately quantifies SMN1 and SMN2 copy numbers to confirm diagnosis and predict phenotypic severity.
Duchenne/Becker Muscular Dystrophy (DMD/BMD): Detects large exonic deletions and duplications in the DMD gene, accounting for 65-75% of pathogenic variants.
Rett Syndrome: Detects large deletions or duplications in the MECP2 gene.
Methylation-Specific MLPA (MS-MLPA): Utilizes probes containing restriction enzyme recognition sites sensitive to DNA methylation to simultaneously detect copy number changes and altered methylation patterns.
Diagnostic tool for Prader-Willi Syndrome, Angelman Syndrome, Beckwith-Wiedemann Syndrome, and Silver-Russell Syndrome.
Microdeletion And Microduplication Syndromes
Evaluates subtelomeric rearrangements, a significant cause of idiopathic intellectual disability.
Detects classical contiguous gene deletion syndromes like DiGeorge Syndrome (22q11.2) and Williams Syndrome (7q11.23).
Advantages And Limitations
Feature
Advantages
Limitations
Diagnostic Scope
High multiplexing capacity (up to 50-60 distinct genomic targets in a single tube).
Targeted approach; detects copy number changes only in specific sequences targeted by the probes.
Resolution Limits
Capable of detecting single exon deletions/duplications, exceeding the resolution of standard FISH or karyotyping.
Cannot detect balanced rearrangements (translocations, inversions), point mutations, intronic variants, or triplet repeats.
Logistics
Cost-effective, high throughput, and requires minute amounts of genomic DNA (as low as 20 ng).
Susceptible to benign Single Nucleotide Polymorphisms (SNPs) at the ligation site, which can falsely indicate a deletion (allele drop-out).
