Abstract
Site-directed mutagenesis is a powerful method to introduce mutation(s) into DNA sequences. A number of methods have been developed over the years with a main goal being to create a high number of mutant genes. The single-mutagenic primer method for site-directed mutagenesis is the most direct method that yields mutant genes in about 25–50 % of transformants in a robust, low-cost reaction. The supercompetent XL10-Gold bacteria used in the Stratagene protocol carry a phage, which may be a problem for some applications; however, in our single-mutagenic primer method the supercompetent bacteria are not needed. A thermostable DNA polymerase with high fidelity and processivity, such as Phusion DNA polymerase, is required for our optimized procedure to avoid extra mutation(s) and enhance mutagenic efficiency.
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Acknowledgments
National Natural Science Foundation of China Grant (No. 41306131), Natural Science Foundation for College and University of Jiangsu Province (No. 13KJB180029), and Provincial Natural Science Foundation of Jiangsu, China grant (No. BK20130440) are acknowledged.
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Huang, Y., Zhang, L. (2017). An In Vitro Single-Primer Site-Directed Mutagenesis Method for Use in Biotechnology. In: Reeves, A. (eds) In Vitro Mutagenesis. Methods in Molecular Biology, vol 1498. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6472-7_26
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DOI: https://doi.org/10.1007/978-1-4939-6472-7_26
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