Global DNA Methylation Detection System Using MBD-Fused Luciferase Based on Bioluminescence Resonance Energy Transfer Assay
| Autor: | Wataru Yoshida, Isao Karube, Yuji Baba |
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| Rok vydání: | 2016 |
| Předmět: |
Models
Molecular 0301 basic medicine Recombinant Fusion Proteins Bisulfite sequencing Biosensing Techniques 01 natural sciences Analytical Chemistry 03 medical and health sciences chemistry.chemical_compound Protein Domains Neoplasms Humans Methylated DNA immunoprecipitation Luciferases Chemistry 010401 analytical chemistry DNA Methylation Molecular biology 0104 chemical sciences DNA-Binding Proteins genomic DNA 030104 developmental biology Energy Transfer CpG site Luminescent Measurements DNA methylation Illumina Methylation Assay CpG Islands Human genome DNA HeLa Cells |
| Zdroj: | Analytical Chemistry. 88:9264-9268 |
| ISSN: | 1520-6882 0003-2700 |
| Popis: | DNA methylation plays an important role in the regulation of gene expression. In normal cells, transposable elements that constitute approximately 45% of the human genome are highly methylated to silence their expression. In cancer cells, transposable elements are hypomethylated; therefore, global DNA methylation level is considered as a biomarker for cancer diagnostics. In this study, a homogeneous assay for measuring global DNA methylation level based on bioluminescence resonance energy transfer (BRET) was developed using methyl-CpG binding domain (MBD)-fused luciferase. In this assay, the MBD-luciferase recognizes methylated CpG, thus, BRET between the luciferase and fluorescent DNA intercalating dye is detected. We demonstrated that the BRET signal depended on the DNA methylation level of the target DNA. Moreover, the BRET signal was correlated with the LINE1 DNA methylation level on human genomic DNA, as determined by the bisulfite method. These results indicate that the global DNA methylation level of human genomic DNA could be detected simply by measuring the BRET signal. |
| Databáze: | OpenAIRE |
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