The Use of Methylation-Sensitive Multiplex Ligation-Dependent Probe Amplification for Quantification of Imprinted Methylation
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.
Key wordsImprinting DNA methylation Methylation-sensitive multiplex ligation-dependent probe amplification
This work was supported by Spanish Ministry of Economy and Competitiveness (MINECO) (BFU2014-53093-R to D.M) cofunded with the European Union Regional Development Fund (FEDER); Institute of Health Carlos III of the Ministry of Economy and Competitiveness cofinanced with European Union ERDF funds (PI13/00467 to G.P.dN.); the Basque Department of Health (GV2014111017 to G.P.dN.) and the I3SNS Program of the Spanish Ministry of Health (CP03/0064; S.I.V.I. 1395/09 to G.P.dN.). AMS is a recipient of an F.P.I. Ph.D. studentship from MINECO. All authors are members of the “European Network for Human Congenital Imprinting Disorders” COST action (BM1208).
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