Abstract
Overlap extension or fusion PCR is thought to be a simple and easy method to produce fusion DNA fragments without the need for restriction enzyme digestion and DNA ligation. However, this method has not been used frequently, probably as it is not always reliable. When natural sequences are used for overlap sequences, sometimes either no fusion DNA is produced or only faint DNA bands are detected owing to low annealing between the overlap sequences selected. Here, we introduce several artificial overlap sequences, most of which are GC-rich, that can be used for reliable fusion PCR. We describe how these overlap sequences can be used for fusion DNA construction, in-frame gene fusion, and cloning in yeast.
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Acknowledgments
The authors thank Sachiko Watanabe, Moemi Yoshiura, Ryota Sakai, and Yukie Misumi for their technical assistance. This work was supported in part by the New Energy and Industrial Technology Development Organization (NEDO) and by the Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN), Japan.
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Cha-aim, K., Hoshida, H., Fukunaga, T., Akada, R. (2012). Fusion PCR via Novel Overlap Sequences. In: Peccoud, J. (eds) Gene Synthesis. Methods in Molecular Biology, vol 852. Humana Press. https://doi.org/10.1007/978-1-61779-564-0_8
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DOI: https://doi.org/10.1007/978-1-61779-564-0_8
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