Citation: Pei Liu, Xiao-Hai Yang, Qing Wang, Jing Huang, Jian-Bo Liu, Ying Zhu, Lei-Liang He, Ke-Min Wang. Sensitive detection of DNA methyltransferase activity based on rolling circle amplifi cation technology[J]. Chinese Chemical Letters, ;2014, 25(7): 1047-1051. doi: 10.1016/j.cclet.2014.05.002 shu

Sensitive detection of DNA methyltransferase activity based on rolling circle amplifi cation technology

1.
State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Key Laboratory for Bio-nanotechnology and Molecular Engineering of Hunan Province, Hunan University, Changsha 410082, China

Corresponding author: Xiao-Hai Yang, Ke-Min Wang,
Received Date: 3 March 2014
Available Online: 14 April 2014
Fund Project: and International Science & Technology operation Program of China (No. 2010DFB30300). (No. 2011CB911002)

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, neithermethylation/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.
- Methyltransferase,
- RCA,
- Dam Mtase,
- Dpn I,
- Fluorescence
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