5.5 ) Multiplex ligation-dependent probe
amplification (MLPA)
Multiplex ligation-dependent probe amplification (MLPA) allows detection of DNA copy number changes of up to 45 loci in one relatively simple, semi quantitative polymerase chain reactionbased assay. Multiplex Ligation-dependent Probe Amplification (MLPA) assay is a recently developed technique able to evidence variations in the copy number of several human genes. Due to this ability, MLPA can be used in the molecular diagnosis of several genetic diseases whose pathogenesis is related to the presence of deletions or duplications of specific genes. Moreover, MLPA assay can also be used in the molecular diagnosis of genetic diseases characterized by the presence of abnormal DNA methylation. Due to the large number of genes that can be analyzed by a single technique, MLPA assay represents the gold standard for molecular analysis of all pathologies derived from the presence of gene copy number variation.
It allows for relative quantification of up to 50 different target sequences in one reaction and does not require living cells or cell culture. It is less labor-intensive and less expensive compared to karyotyping and FISH. Therefore, MLPA has been widely applied for molecular diagnosis of genetic diseases such as DMD, Spinocerebellar ataxia type 15 and chromosomal aneuploidies
Amplification of a probe is made dependent on a ligation step, which can only occur if target DNA is present in the sample. A primary drawback of MLPA is its dependence on length-based discrimination of the ligation products. To differentiate between amplification products, the probes contain a non-hybridizing stuffer sequence of variable length. Therefore, MLPA limits the number of probes to 50 pairs or fewer.