A robust and simple-to-design multiplex DNA methylation assay based on MS-MLPA-CE-SSCP

• DOI: 10.1039/c3an01178j
• Publication Date: 2013-09-09T13:02:09Z
• Authors: Jeongkyeong Na; Gi Won Shin; Gyu Yong Jung; Gyoo Yeol Jung
• Keywords: Multiplex; HpaII; Illumina Methylation Assay

Aberrant DNA methylation is a potential diagnostic marker for complex diseases, such as cancer. With the increase in number of genes known to exhibit disease-associated aberrant methylation, need accurate multiplex assays quantifying has increased. Methylation-specific ligation-dependent probe amplification (MS-MLPA) one method that been highlighted this context. However, two limitations make custom design MS-MLPA impractical: long probes containing stuffer sequences and reliance on only restriction enzyme. Here, we developed variation employs simpler probe-design process. To overcome above-mentioned limitations, used stuffer-free are subsequently analyzed using high-resolution capillary electrophoresis-based single-strand conformational polymorphism (CE-SSCP) instead conventional length-dependent CE. Moreover, multiple methylation-sensitive enzymes (HhaI, HpaII, AciI) were simultaneously; thus, satisfying desired criteria available all targets. Using assay concept, 17 associated with hepatocellular carcinoma. Our results showed custom-designed based MS-MLPA-CE-SSCP provided robust quantification levels.