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Updated: Jun 4, 2026

Site-Directed Mutagenesis for In Vitro and In Vivo Experiments Exemplified with RNA Interactions in Escherichia Coli
Published on: February 5, 2019
A "mesmer"izing new approach to site-directed mutagenesis in large transformation-ready constructs: Mutagenesis via
Julie S Jacobs1, Xiaojing Hong, Daniel F Eberl
1Department of Biology, University of Iowa, Iowa City, IA, USA.
Abstract:
Creating designer mutations in large genes is a challenge. Size limitations imposed by site-directed mutagenesis (SDM), coupled with the paucity of unique restriction enzyme sites, make subsequent cloning of these constructs extremely difficult. "Mutagenesis via Serial Small Mismatch Recombineering" (MSSMR) combines sequential recombineering steps with SDM to create seamless, pre-specified mutations as small as a single base pair. We demonstrate the simultaneous cloning of wild type and mutant constructs of a > 30 kb gene directly into attB transformation vectors. No post-transformation manipulations are required, and because the technique relies on recombineering methods, addition of undesired mutations via PCR is minimized.
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