Abstract
Here we present a simple and convenient sticky/blunt-end ligation method for fusion gene construction. The fusion gene is constructed by seamless ligation of 5′-end phosphorylated blunt ends instead of by overlap extension PCR (OE-PCR). Therefore, the challenge of amplifying large DNA fragments (e.g., the large bifunctional enzyme gene constructed by fusion of two monofunctional enzyme genes) by PCR can be avoided. In addition, synthesis of the inner primers for OE-PCR is not necessary, indicating that this method should be especially convenient for construction of fusion genes with various combinations of multiple fragments (e.g., chimeric gene libraries, fusion gene libraries). As a modification of the commonly used fusion gene construction technique, this method may find a wide range of applications in bioscience and biotechnology.
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Acknowledgments
This work was supported by the National Natural Science Foundations of China (nos. 31100045 and 31270114), Program for Liaoning Excellent Talents in the University (no. LJQ2011067) and the Natural Science Foundation of Shandong Province (no. ZR2013BM001).
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Song Gao and Jiannan Zhang have contributed equally to this work.
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Gao, S., Zhang, J., Miao, T. et al. A Simple and Convenient Sticky/Blunt-End Ligation Method for Fusion Gene Construction. Biochem Genet 53, 42–48 (2015). https://doi.org/10.1007/s10528-015-9669-x
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DOI: https://doi.org/10.1007/s10528-015-9669-x