Journal
CHINESE CHEMICAL LETTERS
Volume 25, Issue 7, Pages 1047-1051Publisher
ELSEVIER SCIENCE INC
DOI: 10.1016/j.cclet.2014.05.002
Keywords
Methyltransferase; RCA; Dam MTase; Dpn I; Fluorescence
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Funding
- National Natural Science Foundation of China [21190044, 21175035]
- National Basic Research Program [2011CB911002]
- International Science & Technology operation Program of China [2010DFB30300]
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This work develops a fluorescence approach for sensitive detection of DNA methyltransferase activity based on endonuclease and rolling circle amplification (RCA) technique. In the presence of DNA adenine methylation (Dam) MTase, the methylation-responsive sequence of hairpin probe is methylated and cleaved by the methylation-sensitive restriction endonuclease Dpn I. The products cleaved by restriction endonuclease Dpn I then function as a signal primer to initiate RCA reaction by hybridizing with the circular DNA template. Each RCA product containing thousands of repeated sequences might hybridize with a large number of molecular beacons (detection probes), resulting in an enhanced fluorescence signal. In the absence of Dam MTase, neither methylation/cleavage nor RCA reaction can be initiated and no fluorescence signal is observed. The proposed method exhibits a dynamic range from 0.5 U/mL to 30 U/mL and a detection limit of 0.18 U/mL. This method can be used for the screening of antimicrobial drugs and has a great potential to be further applied in early clinical diagnosis. (C) 2014 Xiao-Hai Yang and Ke-Min Wang. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
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