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.
Preview |
Text
Montanaro_journal.pone.0177788.pdf - Published Version Download (4MB) | Preview |
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.
| Type: | Article |
|---|---|
| Title: | Efficient method for site-directed mutagenesis in large plasmids without subcloning |
| Location: | United States |
| Open access status: | An open access version is available from UCL Discovery |
| DOI: | 10.1371/journal.pone.0177788 |
| Publisher version: | http://dx.doi.org/10.1371/journal.pone.0177788 |
| Language: | English |
| Additional information: | © 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. |
| UCL classification: | UCL UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Population Health Sciences > UCL GOS Institute of Child Health UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Population Health Sciences > UCL GOS Institute of Child Health > Developmental Neurosciences Dept |
| URI: | https://discovery.ucl.ac.uk/id/eprint/1559273 |
Loading...
Loading...Loading...Loading...Archive Staff Only
 |
View Item |
