Skip navigation
Skip navigation

Mutant T4 DNA polymerase for easy cloning and mutagenesis

Qi, Ruhu; Otting, Gottfried

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.authorQi, Ruhu
dc.contributor.authorOtting, Gottfried
dc.date.accessioned2019-01-28T23:37:46Z
dc.date.available2019-01-28T23:37:46Z
dc.identifier.issn1932-6203
dc.identifier.urihttp://hdl.handle.net/1885/155277
dc.description.abstractThe 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.mimetypeapplication/pdf
dc.format.mimetypeapplication/pdf
dc.language.isoen_AU
dc.publisherPublic Library of Science
dc.rightsCopyright: © 2019 Qi, Otting.
dc.sourcePLOS ONE
dc.titleMutant T4 DNA polymerase for easy cloning and mutagenesis
dc.typeJournal article
local.description.notesImported from PLOS
local.description.notesImported from PLOS
dc.date.issued2019
local.identifier.ariespublicationu3102795xPUB661
local.type.statusPublished Version
local.type.statusPublished Version
local.contributor.affiliationOtting, G., Research School of Chemistry, The Australian National University
dc.relationhttp://purl.org/au-research/grants/arc/DP170100162
dc.relationhttp://purl.org/au-research/grants/arc/FL170100019
local.identifier.doi10.1371/journal.pone.0211065
dc.date.updated2019-01-27T09:05:29Z
dcterms.accessRightsOpen Access
dc.provenanceThis 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.
CollectionsANU Research Publications

Download

File Description SizeFormat Image
journal.pone.0211065.pdf949.23 kBAdobe PDFThumbnail


Items in Open Research are protected by copyright, with all rights reserved, unless otherwise indicated.

Updated:  17 November 2022/ Responsible Officer:  University Librarian/ Page Contact:  Library Systems & Web Coordinator