MLPA

MLPA, or multiplex ligation-dependent probe amplification, is a molecular biology technique used to detect abnormal copy numbers of DNA sequences, particularly exon-level deletions and duplications across one or more genes. The method is designed to be high-throughput and cost-effective for targeted panels of genomic regions.

The principle relies on designed probe pairs that hybridize adjacent to each other on the target DNA. Each probe consists of two parts that bind side by side; if both parts hybridize correctly, a DNA ligase joins them to form a single probe that serves as a template for PCR. The ligated probes share a universal primer sequence, allowing all targets to be amplified simultaneously in a single PCR reaction. Each probe pair is assigned a specific length, so the resulting amplified products can be separated and quantified by capillary electrophoresis. The measured peak heights or areas reflect the amount of target sequence present, enabling relative copy-number estimation after normalization against reference samples and internal control probes.

Workflow typically involves extracting genomic DNA, hybridizing a commercially prepared probe mix, performing ligation, amplifying with fluorescent universal primers, and analyzing the mixture by capillary electrophoresis. Data are interpreted by comparing peak intensities to those from normal reference samples to determine deletions or duplications in the targeted regions.

MLPA is widely used in clinical genetics and research to screen for exon-level copy-number variations in genes associated with hereditary disorders, chromosomal abnormalities, and certain cancers. Advantages include multiplexing capability, relatively low cost, and tolerance for somewhat degraded DNA. Limitations include reliance on known target sequences, potential sensitivity to single-nucleotide variants at probe binding sites, and dependence on careful normalization; balanced rearrangements are not detected by MLPA.