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Intra-Molecular Homologous Recombination of Scarless Plasmid

Although many methods have been reported, plasmid construction compromises transformant efficiency (number of transformants per ng of DNAs) with plasmid accuracy (rate of scarless plasmids). An efficient method is two-step PCR serving DNA amplification. An accurate method is ExnaseII cloning serving...

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Autores principales: Liang, Yaping, Zhang, Yu, Liu, Liangwei
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7084384/
https://www.ncbi.nlm.nih.gov/pubmed/32131382
http://dx.doi.org/10.3390/ijms21051697
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author Liang, Yaping
Zhang, Yu
Liu, Liangwei
author_facet Liang, Yaping
Zhang, Yu
Liu, Liangwei
author_sort Liang, Yaping
collection PubMed
description Although many methods have been reported, plasmid construction compromises transformant efficiency (number of transformants per ng of DNAs) with plasmid accuracy (rate of scarless plasmids). An efficient method is two-step PCR serving DNA amplification. An accurate method is ExnaseII cloning serving homology recombination (HR). We combine DNA amplification and HR to develop an intra-molecular HR by amplifying plasmid DNAs to contain homology 5′- and 3′-terminus and recombining the plasmid DNAs in vitro. An example was to construct plasmid pET20b-AdD. The generality was checked by constructing plasmid pET21a-AdD and pET22b-AdD in parallel. The DNAs having 30-bp homology arms were optimal for intra-molecular HR, and transformation of which created 14.2 transformants/ng and 90% scarless plasmids, more than the two-step PCR and the ExnaseII cloning. Transformant efficiency correlated with the component of nicked circular plasmid DNAs of HR products, indicating nick modification in vivo leads to scar plasmids.
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spelling pubmed-70843842020-03-24 Intra-Molecular Homologous Recombination of Scarless Plasmid Liang, Yaping Zhang, Yu Liu, Liangwei Int J Mol Sci Article Although many methods have been reported, plasmid construction compromises transformant efficiency (number of transformants per ng of DNAs) with plasmid accuracy (rate of scarless plasmids). An efficient method is two-step PCR serving DNA amplification. An accurate method is ExnaseII cloning serving homology recombination (HR). We combine DNA amplification and HR to develop an intra-molecular HR by amplifying plasmid DNAs to contain homology 5′- and 3′-terminus and recombining the plasmid DNAs in vitro. An example was to construct plasmid pET20b-AdD. The generality was checked by constructing plasmid pET21a-AdD and pET22b-AdD in parallel. The DNAs having 30-bp homology arms were optimal for intra-molecular HR, and transformation of which created 14.2 transformants/ng and 90% scarless plasmids, more than the two-step PCR and the ExnaseII cloning. Transformant efficiency correlated with the component of nicked circular plasmid DNAs of HR products, indicating nick modification in vivo leads to scar plasmids. MDPI 2020-03-02 /pmc/articles/PMC7084384/ /pubmed/32131382 http://dx.doi.org/10.3390/ijms21051697 Text en © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Liang, Yaping
Zhang, Yu
Liu, Liangwei
Intra-Molecular Homologous Recombination of Scarless Plasmid
title Intra-Molecular Homologous Recombination of Scarless Plasmid
title_full Intra-Molecular Homologous Recombination of Scarless Plasmid
title_fullStr Intra-Molecular Homologous Recombination of Scarless Plasmid
title_full_unstemmed Intra-Molecular Homologous Recombination of Scarless Plasmid
title_short Intra-Molecular Homologous Recombination of Scarless Plasmid
title_sort intra-molecular homologous recombination of scarless plasmid
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7084384/
https://www.ncbi.nlm.nih.gov/pubmed/32131382
http://dx.doi.org/10.3390/ijms21051697
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AT zhangyu intramolecularhomologousrecombinationofscarlessplasmid
AT liuliangwei intramolecularhomologousrecombinationofscarlessplasmid