Detection and discrimination of maintenance and de novo CpG methylation events using MethylBreak.
Biosens Bioelectron
; 91: 658-663, 2017 May 15.
Article
en En
| MEDLINE
| ID: mdl-28110250
ABSTRACT
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.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
ADN
/
Técnicas Biosensibles
/
Islas de CpG
/
Metilación de ADN
/
ADN (Citosina-5-)-Metiltransferasas
Tipo de estudio:
Diagnostic_studies
/
Prognostic_studies
Límite:
Humans
Idioma:
En
Revista:
Biosens Bioelectron
Asunto de la revista:
BIOTECNOLOGIA
Año:
2017
Tipo del documento:
Article
País de afiliación:
Taiwán