Sensitive detection of DNA methyltransferase activity based on rolling circle amplification technology  

作　　者：Pei Liu Xiao-Hai Yang Qing Wang Jing Huang Jian-Bo Liu Ying Zhu Lei-Liang He Ke-Min Wang 

机构地区：[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

出　　处：《Chinese Chemical Letters》2014年第7期1047-1051,共5页中国化学快报（英文版）

基　　金：supported by the National Natural Science Foundation of China(Nos.21190044 and 21175035);National Basic Research Program(No.2011CB911002);International Science&Technology operation Program of China(No.2010DFB30300)

摘　　要：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 1. 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.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 1. 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.

关 键 词：METHYLTRANSFERASE RCAD am MTase Dpn 1 FLUORESCENCE 

分 类 号：O657.3[理学—分析化学] O629.8[理学—化学]