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Efficient One-Step Fusion PCR Based on Dual-Asymmetric Primers and Two-Step Annealing

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Abstract

Gene splicing by fusion PCR is a versatile and widely used methodology, especially in synthetic biology. We here describe a rapid method for splicing two fragments by one-round fusion PCR with a dual-asymmetric primers and two-step annealing (ODT) method. During the process, the asymmetric intermediate fragments were generated in the early stage. Thereafter, they were hybridized in the subsequent cycles to serve as template for the target full-length product. The process parameters such as primer ratio, elongation temperature and cycle numbers were optimized. In addition, the fusion products produced with this method were successfully applied in seamless genome editing. The fusion of two fragments by this method takes less than 0.5 day. The method is expected to facilitate various kinds of complex genetic engineering projects with enhanced efficiency.

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Acknowledgements

This work was supported by the National Basic Research Program (973 Program, 2011CBA00800).

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Authors and Affiliations

  1. Department of Chemical and Biomolecular Engineering, University of Nebraska Lincoln, Lincoln, NE, 68588, USA

    Yilan Liu

  2. State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing, 100190, People’s Republic of China

    Jinjin Chen

  3. Department of Food Science and Technology, University of Nebraska Lincoln, Lincoln, NE, 68588, USA

    Jinjin Chen

  4. Center of Bioprocess Engineering, Department of Chemical and Biochemical Engineering, Technical University of Denmark, 2800, Lyngby, Denmark

    Anders Thygesen

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  1. Yilan Liu
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  2. Jinjin Chen
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  3. Anders Thygesen
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Correspondence to Yilan Liu.

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Liu, Y., Chen, J. & Thygesen, A. Efficient One-Step Fusion PCR Based on Dual-Asymmetric Primers and Two-Step Annealing. Mol Biotechnol 60, 92–99 (2018). https://doi.org/10.1007/s12033-017-0050-7

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  • DOI: https://doi.org/10.1007/s12033-017-0050-7

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