eprintid: 1559273 rev_number: 27 eprint_status: archive userid: 608 dir: disk0/01/55/92/73 datestamp: 2017-06-10 19:11:35 lastmod: 2022-01-03 00:11:08 status_changed: 2017-06-23 12:19:14 type: article metadata_visibility: show creators_name: Hallak, LK creators_name: Berger, K creators_name: Kaspar, R creators_name: Kwilas, AR creators_name: Montanaro, F creators_name: Peeples, ME title: Efficient method for site-directed mutagenesis in large plasmids without subcloning ispublished: pub divisions: UCL divisions: B02 divisions: D13 divisions: G26 note: © 2017 Hallak et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. abstract: Commonly used methods for site-directed DNA mutagenesis require copying the entire target plasmid. These methods allow relatively easy modification of DNA sequences in small plasmids but become less efficient and faithful for large plasmids, necessitating full sequence verification. Introduction of mutations in larger plasmids requires subcloning, a slow and labor-intensive process, especially for multiple mutations. We have developed an efficient DNA mutagenesis technique, UnRestricted Mutagenesis and Cloning (URMAC) that replaces subcloning steps with quick biochemical reactions. URMAC does not suffer from plasmid size constraints and allows simultaneous introduction of multiple mutations. URMAC involves manipulation of only the mutagenesis target site(s), not the entire plasmid being mutagenized, therefore only partial sequence verification is required. Basic URMAC requires two PCR reactions, each followed by a ligation reaction to circularize the product, with an optional third enrichment PCR step followed by a traditional cloning step that requires two restriction sites. Here, we demonstrate URMAC's speed, accuracy, and efficiency through several examples, creating insertions, deletions or substitutions in plasmids ranging from 2.6 kb to 17 kb without subcloning. date: 2017 date_type: published official_url: http://dx.doi.org/10.1371/journal.pone.0177788 oa_status: green full_text_type: pub pmcid: PMC5456045 language: eng primo: open primo_central: open_green article_type_text: Journal Article verified: verified_manual elements_id: 1297874 doi: 10.1371/journal.pone.0177788 pii: PONE-D-16-51638 lyricists_name: Montanaro, Federica lyricists_id: FMONT29 actors_name: Dewerpe, Marie actors_id: MDDEW97 actors_role: owner full_text_status: public publication: PLoS One volume: 12 number: 6 article_number: e0177788 event_location: United States issn: 1932-6203 citation: Hallak, LK; Berger, K; Kaspar, R; Kwilas, AR; Montanaro, F; Peeples, ME; (2017) Efficient method for site-directed mutagenesis in large plasmids without subcloning. PLoS One , 12 (6) , Article e0177788. 10.1371/journal.pone.0177788 <https://doi.org/10.1371/journal.pone.0177788>. Green open access document_url: https://discovery.ucl.ac.uk/id/eprint/1559273/1/Montanaro_journal.pone.0177788.pdf