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Research Article

Academia Journal of Biotechnology 6(3): 062-065, March 2018
DOI: 10.15413/ajb.2018.0106
ISSN: 2315-7747
©2018 Academia Publishing

Abstract


Quick inactivation of BglI sites in pUC18 using in-fusion® mediated site-directed mutagenesis
 

Accepted 20th March, 2018
 
Hongmin Li*, Jihong Cui, Shasha Yang, Sili Wei, Xin Xie and Fulin Chen

College of Life Sciences, Northwest University, Key Laboratory of Resource Biology and Biotechnology in Western China, Ministry of China, Taibai North Rd 229, 710069, Xi’an, Shaanxi Province, China.

 

In-Fusion® strategy is a highly regarded strategy in gene cloning manipulation. Here, we reported a successful case of site-directed mutagenesis mediated by In-Fusion® strategy. The 1118 bp fragment between the two sites of BglI in pUC18 was amplified using the primers containing mutation site and recombined with the 1568 bp fragments of pUC18 derived from the digestion of BglI in virtue of the 15 bp homologous sequence added to the 5’-end of each primer that precisely match the ends of the 1568 bp fragment. Restriction digestion and DNA sequencing showed that the plasmids isolated from the 6 single colonies selected randomly were successfully mutated with the BglI436 replaced by XhoI436 and inactive BglI1554, indicating mutation efficiency in this work was very high and the manipulation was simpler than other means as the fragment containing mutation site was not required to be treated by restriction endonuclease before recombination. Furthermore, the cost of this technique was lower than gene synthesis when designed mutation site was just only a base, a site of restriction endonuclease or other short elements. These results proved that In-Fusion® cloning technique can be used as an effective approach for site-specific mutagenesis.

Keywords: Quick-mutation, gene cloning, recombinant, elastin-like protein.

 

This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Cite this article as:
Li H, Cui J, Yang S, Wei S, Xie X, Chen F (2018). Quick inactivation of BglI sites in pUC18 using in-fusion® mediated site-directed mutagenesis. Acad. J. Biotechnol. 6(3): 062-065.

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