TY - JOUR
T1 - Detection and discrimination of maintenance and de novo CpG methylation events using MethylBreak
AU - Hsu, William
AU - Mercado, Augustus T.
AU - Hsiao, George
AU - Yeh, Jui Ming
AU - Chen, Chung Yung
PY - 2017/5/15
Y1 - 2017/5/15
N2 - Understanding the principles governing the establishment and maintenance activities of DNA methyltransferases (DNMTs) can help in the development of predictive biomarkers associated with genetic disorders and diseases. A detection system was developed that distinguishes and quantifies methylation events using methylation-sensitive endonucleases and molecular beacon technology. MethylBreak (MB) is a 22-mer oligonucleotide with one hemimethylated and two unmethylated CpG sites, which are also recognition sites for Sau96I and SacII, and is attached to a fluorophore and a quencher. Maintenance methylation was quantified by fluorescence emission due to the digestion of SacII when the hemimethylated CpG site is methylated, which inhibits Sau96I cleavage. The signal difference between SacII digestion of both MB substrate and maintenance methylated MB corresponds to de novo methylation event. Our technology successfully discriminated and measured both methylation activities at different concentrations of MB and achieved a high correlation coefficient of R2=0.997. Additionally, MB was effectively applied to normal and cancer cell lines and in the analysis of enzymatic kinetics and RNA inhibition of recombinant human DNMT1.
AB - Understanding the principles governing the establishment and maintenance activities of DNA methyltransferases (DNMTs) can help in the development of predictive biomarkers associated with genetic disorders and diseases. A detection system was developed that distinguishes and quantifies methylation events using methylation-sensitive endonucleases and molecular beacon technology. MethylBreak (MB) is a 22-mer oligonucleotide with one hemimethylated and two unmethylated CpG sites, which are also recognition sites for Sau96I and SacII, and is attached to a fluorophore and a quencher. Maintenance methylation was quantified by fluorescence emission due to the digestion of SacII when the hemimethylated CpG site is methylated, which inhibits Sau96I cleavage. The signal difference between SacII digestion of both MB substrate and maintenance methylated MB corresponds to de novo methylation event. Our technology successfully discriminated and measured both methylation activities at different concentrations of MB and achieved a high correlation coefficient of R2=0.997. Additionally, MB was effectively applied to normal and cancer cell lines and in the analysis of enzymatic kinetics and RNA inhibition of recombinant human DNMT1.
KW - de novo methylation
KW - DNA methyltransferases
KW - Maintenance methylation
KW - Methylation-sensitive endonucleases
KW - MethylBreak
KW - Molecular beacon
UR - http://www.scopus.com/inward/record.url?scp=85010014422&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85010014422&partnerID=8YFLogxK
U2 - 10.1016/j.bios.2017.01.026
DO - 10.1016/j.bios.2017.01.026
M3 - Article
C2 - 28110250
AN - SCOPUS:85010014422
SN - 0956-5663
VL - 91
SP - 658
EP - 663
JO - Biosensors and Bioelectronics
JF - Biosensors and Bioelectronics
ER -