Mutant T4 DNA polymerase for easy cloning and mutagenesis
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Description
The advent of high-fidelity DNA polymerases that can be used to linearize and amplify whole plasmids by PCR opened the door to greatly simplified cloning and mutagenesis protocols. Commercially available kits work well, but often have been optimized using undisclosed or proprietory components. Here we show that a mutant T4 DNA polymerase (Y320A) with attenuated 3'-exonuclease activity is uniquely suited to generate single-stranded DNA overhangs of uniform length in a more easily controllable...[Show more]
dc.contributor.author | Qi, Ruhu | |
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dc.contributor.author | Otting, Gottfried | |
dc.date.accessioned | 2019-01-28T23:37:46Z | |
dc.date.available | 2019-01-28T23:37:46Z | |
dc.identifier.issn | 1932-6203 | |
dc.identifier.uri | http://hdl.handle.net/1885/155277 | |
dc.description.abstract | The advent of high-fidelity DNA polymerases that can be used to linearize and amplify whole plasmids by PCR opened the door to greatly simplified cloning and mutagenesis protocols. Commercially available kits work well, but often have been optimized using undisclosed or proprietory components. Here we show that a mutant T4 DNA polymerase (Y320A) with attenuated 3'-exonuclease activity is uniquely suited to generate single-stranded DNA overhangs of uniform length in a more easily controllable manner than the wild-type enzyme, and this can be used to increase the yields of colonies containing correctly modified plasmids in cloning and mutagenesis experiments, which is particularly useful when E. coli cells are of relatively low competency. Standard protocols using the mutant T4 DNA polymerase are provided for the sequence and ligation independent cloning (SLIC) method and a modified QuikChange method, where the mutant enzyme enhances the yield of correctly mutated plasmid and further suppresses parental plasmid during digestion with DpnI. Single-stranded DNA overhangs generated by the mutant T4 DNA polymerase facilitate subsequent plasmid circularization, annealing and ligation in E. coli. | |
dc.format.mimetype | application/pdf | |
dc.format.mimetype | application/pdf | |
dc.language.iso | en_AU | |
dc.publisher | Public Library of Science | |
dc.rights | Copyright: © 2019 Qi, Otting. | |
dc.source | PLOS ONE | |
dc.title | Mutant T4 DNA polymerase for easy cloning and mutagenesis | |
dc.type | Journal article | |
local.description.notes | Imported from PLOS | |
local.description.notes | Imported from PLOS | |
dc.date.issued | 2019 | |
local.identifier.ariespublication | u3102795xPUB661 | |
local.type.status | Published Version | |
local.type.status | Published Version | |
local.contributor.affiliation | Otting, G., Research School of Chemistry, The Australian National University | |
dc.relation | http://purl.org/au-research/grants/arc/DP170100162 | |
dc.relation | http://purl.org/au-research/grants/arc/FL170100019 | |
local.identifier.doi | 10.1371/journal.pone.0211065 | |
dc.date.updated | 2019-01-27T09:05:29Z | |
dcterms.accessRights | Open Access | |
dc.provenance | 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. | |
Collections | ANU Research Publications |
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