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An isothermal strand displacement amplification strategy for nucleic acids using junction forming probes and colorimetric detection

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Abstract

The authors describe a method for DNA target recognition and signal amplification that is based on the target-induced formation of a three way junction. The subsequent assembly of two DNA probes releases the inhibitory strand and triggers a downstream strand displacement amplification. This causes the formation of a G-rich single sequence that binds to a hemin monomer with its peroxidase-mimicking properties. The resulting peroxidase (POx) activity is quantified by using H2O2 and TMB as the substrate. In the presence of an inhibitor, in contrast, the POx-like activity is strongly reduced. This forms the basis for a highly sensitive DNA assay. It has a 0.8 pM detection limit when operated at a wavelength of 450 nm and was applied to the isothermal determination of target DNA with high selectivity.

Schematic of the assay: Introduction of target results in the formation of a three way junction. The subsequent assembly of two probes releases the inhibitory strand and triggers a downstream strand displacement amplification, generating amount of G-rich single sequence which causes peroxidase-mimicking activity on binding to a hemin monomer.

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Acknowledgements

This work was supported by the National Natural Science Foundation of China (Grant nos. 81271917), Natural Science Foundation of Zhejiang province (Grant nos. LY14H200002, LY15H200002 and LY16H160023). We thank Clinical Research Centre from the Second Affiliated Hospital of Zhejiang University School of Medicine for essential technical supports.

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Authors and Affiliations

  1. Department of Laboratory Medicine, the Second Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou, China

    Xuchu Wang, Weiwei Liu, Binbin Yin, Yiwen Sang, Zhenping Liu, Yu Dai, Xiuzhi Duan, Gong Zhang & Zhihua Tao

  2. Key Laboratory of Clinical Laboratory Diagnostics (Ministry of Education), College of Laboratory Medicine, Chongqing Medical University, Chongqing, 400016, China

    Shijia Ding

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  1. Xuchu Wang
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  2. Weiwei Liu
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  3. Binbin Yin
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  4. Yiwen Sang
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  9. Shijia Ding
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  10. Zhihua Tao
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The author(s) declare that they have no competing interests.

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Xuchu Wang and Weiwei Liu contributed equally to this work.

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Wang, X., Liu, W., Yin, B. et al. An isothermal strand displacement amplification strategy for nucleic acids using junction forming probes and colorimetric detection. Microchim Acta 184, 1603–1610 (2017). https://doi.org/10.1007/s00604-017-2158-7

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  • DOI: https://doi.org/10.1007/s00604-017-2158-7

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