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Identification of CpG methylation of the SNRPN gene by methylation‐specific multiplex PCR
Author(s) -
Hung ChiaCheng,
Lin ShinYu,
Lin ShuanPei,
Niu DouMing,
Lee NiChung,
Cheng WenFang,
Chen ChihPing,
Lin WinLi,
Lee ChienNan,
Su YiNing
Publication year - 2009
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.200800225
Subject(s) - methylation , illumina methylation assay , multiplex , bisulfite sequencing , biology , cpg site , dna methylation , methylated dna immunoprecipitation , microbiology and biotechnology , multiplex polymerase chain reaction , genetics , epigenetics , gene , polymerase chain reaction , gene expression
In this article, we show that methylation‐specific multiplex PCR (MS‐multiplex PCR) is a sensitive and specific single assay for detecting CpG methylation status as well as copy number aberrations. We used MS‐multiplex PCR to simultaneously amplify three sequences: the 3′ ends of the SNRPN gene (for unmethylated sequences), the KRITI gene (as internal control), and the promoter of the SNRPN gene containing CpG islands (for methylated sequences) after digestion with a methylation‐sensitive restriction enzyme ( Hha I). We established this duplex assay for the analysis of 38 individuals with Prader–Willi syndrome, 2 individuals with Angelman syndrome, and 28 unaffected individuals. By comparing the copy number of the three regions, the methylation status and the copy number changes can be easily distinguished by MS‐multiplex PCR without the need of bisulfite treatment of the DNA. The data showed that MS‐multiplex PCR allows for the estimation of the methylation level by comparing the copy number aberrations of unknown samples to the standards with a known methylated status. The in‐house‐designed MS‐multiplex PCR protocol is a relatively simple, cost‐effective, and highly reproducible approach as a significant strategy in clinical applications for epigenetics in a routine laboratory.