The MLPA (multiplex ligation-dependent probe amplification) method for detecting dosage differences of up to 50 different nucleic acid fragments in a single reaction was first described in 2002 (Schouten et al., Nucl Acids Res 30:12e57). In this study, the copy number of 40 loci in the human genome was determined through simultaneous relative quantification. The amount of sequence-specific hybridised and ligated oligonucleotides is proportional to the copy number of the target sequence.

The amplification products are subsequently separated by size using capillary electrophoresis. Dose differences are identified by reductions or increases in heights and areas of the detected peaks.

To date, more than 300 different MLPA kits are commercially available for the detection of deletions/duplications of single exons within genes, gene regions, entire genes, chromosomal segments, and even whole chromosomes (e.g., from MRC-Holland).

Variants of the basic MLPA technique have also been developed:

• RT-MLPA (reverse transcriptase MLPA): Used for mRNA profiling.
• MS-MLPA (): Determines copy number and analyses methylation patterns, enabling imprinting defect detection and tumour analysis.