A “mesmer”izing new approach to site-directed mutagenesis in large transformation-ready constructs utagenesis via

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” (...

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Bibliographic Details
Published in:Fly (Austin, Tex.) Vol. 5; no. 2; pp. 162 - 169
Main Authors: Jacobs, Julie S, Hong, Xiaojing, Eberl, Daniel F
Format: Journal Article
Language:English
Published: Landes Bioscience 01-01-2011
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Summary: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.
ISSN:1933-6934
1933-6942
DOI:10.4161/fly.5.2.15092