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

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

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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

    • 通讯作者: Xiao-Hai Yang,
    Ke-Min Wang,
  • 基金项目:

    National Basic Research Program (No. 2011CB911002) (No. 2011CB911002)

    International Science & Technology operation Program of China (No. 2010DFB30300). (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 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.

English

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  • 收稿日期:  2014-03-03
  • 网络出版日期:  2014-04-14
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