Identification of CpG methylation of the SNRPN gene by methylation-specific multiplex PCR.
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 (HhaI). 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.
Hung
Chia-Cheng
CC
Institute of Biomedical Engineering, National Taiwan University, Taipei, Taiwan.
Lin
Shin-Yu
SY
Lin
Shuan-Pei
SP
Niu
Dou-Ming
DM
Lee
Ni-Chung
NC
Cheng
Wen-Fang
WF
Chen
Chih-Ping
CP
Lin
Win-Li
WL
Lee
Chien-Nan
CN
Su
Yi-Ning
YN
D017204:Angelman Syndrome / Q000175:diagnosis; D018899:CpG Islands; D019175:DNA Methylation; D006358:Hot Temperature; D006801:Humans; D016133:Polymerase Chain Reaction / Q000191:economics / Q000379:methods*; D011218:Prader-Willi Syndrome / Q000175:diagnosis; D015203:Reproducibility of Results; D055517:snRNP Core Proteins / Q000235:genetics*
D055517:snRNP Core Proteins