Imprinting disorders are a group of congenital diseases that can result from multiple mechanisms affecting imprinted gene dosage including cytogenetic aberration and epigenetic anomalies. Quantification of CpG methylation and correct copy-number calling is required for molecular diagnosis. Methylation-sensitive multiplex ligation-dependent probe amplification (MS-MLPA) is a multiplex method that accurately measures both parameters in a single assay. This technique relies upon the ligation of MLPA probe oligonucleotides and digestion of the genomic DNA–probe hybrid complexes with the Hha1 methylation-sensitive restriction endonuclease prior to fluorescent PCR amplification with a single primer pair. Since each targeted probe contains stuffer sequence of varying length, each interrogated position is visualized as an amplicon of different size upon capillary electrophoresis.
Monteagudo-Sánchez, A., Garin, I., de Nanclares, G. P., & Monk, D. (2018). The use of methylation-sensitive multiplex ligation-dependent probe amplification for quantification of imprinted methylation. In Methods in Molecular Biology (Vol. 1766, pp. 109–121). Humana Press Inc. https://doi.org/10.1007/978-1-4939-7768-0_6