Hairpin fluorescence DNA probe for real-time monitoring of DNA methylation.
DNA methylation catalyzed by methylase plays an important role in many biological events. However, traditional methods of methylase activity analysis by gel electrophoresis were laborious and discontinuous. In this paper, we report a new strategy to study methylase activity using fluorescent probes coupled with enzyme-linkage reactions. A hairpin DNA probe is prepared with a fluorophore and a quencher linked at the 5'- and 3'-terminus of the probe. A disturbance of the stem sequence by DNA methylation would cause the separation of the fluorophore and the quencher, resulting in the restoration of the fluorescence. We used DNA adenine methylation (Dam) methyltransferase (MTase) and Dpn I endonuclease, both having a 5'-G-A-T-C-3' recognition sequence. Dam MTase catalyzed the methylation of the sequence of 5'-GATC-3', and Dpn I cut the sequence of 5'-G-Am-T-C-3'. The fluorescence of the hairpin probe was restored when it was cleaved by Dpn I endonuclease during the course of methylation. Unlike traditional methods, this assay was done in real time and could be used to monitor the dynamic process of methylation. Our method is easy, simple, and nonradioactive, yet as efficient as gel electrophoresis in detecting the activity of methylase. It also had the potential to screen suitable inhibitor drugs for Dam methylase.
Li
Jun
J
Biomedical Engineering Center, State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry & Chemical Engineering, Hunan University, Changsha 410082, P R China.
Yan
Hongfei
H
Wang
Kemin
K
Tan
Weihong
W
Zhou
Xingwang
X
D019175:DNA Methylation; D015254:DNA Modification Methylases / Q000378:metabolism*; D015342:DNA Probes / Q000737:chemistry*; D015252:Deoxyribonucleases, Type II Site-Specific; D005453:Fluorescence; D005456:Fluorescent Dyes; D008722:Methods; D009690:Nucleic Acid Conformation; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific)
D015342:DNA Probes; D005456:Fluorescent Dyes; D015254:DNA Modification Methylases; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific); C020102:endodeoxyribonuclease DpnI; D015252:Deoxyribonucleases, Type II Site-Specific