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Golden GATEway Cloning – A Combinatorial Approach to Generate Fusion and Recombination Constructs

The design and generation of DNA constructs is among the necessary but generally tedious tasks for molecular biologists and, typically, the cloning strategy is restricted by available restriction sites. However, increasingly sophisticated experiments require increasingly complex DNA constructs, with...

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Autores principales: Kirchmaier, Stephan, Lust, Katharina, Wittbrodt, Joachim
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2013
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3792108/
https://www.ncbi.nlm.nih.gov/pubmed/24116091
http://dx.doi.org/10.1371/journal.pone.0076117
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author Kirchmaier, Stephan
Lust, Katharina
Wittbrodt, Joachim
author_facet Kirchmaier, Stephan
Lust, Katharina
Wittbrodt, Joachim
author_sort Kirchmaier, Stephan
collection PubMed
description The design and generation of DNA constructs is among the necessary but generally tedious tasks for molecular biologists and, typically, the cloning strategy is restricted by available restriction sites. However, increasingly sophisticated experiments require increasingly complex DNA constructs, with an intricacy that exceeds what is achievable using standard cloning procedures. Many transgenes such as inducible gene cassettes or recombination elements consist of multiple components that often require precise in-frame fusions. Here, we present an efficient protocol that facilitates the generation of these complex constructs. The golden GATEway cloning approach presented here combines two established cloning methods, namely golden Gate cloning and Multisite Gateway(TM) cloning. This allows efficient and seamless assembly as well as reuse of predefined DNA elements. The golden Gate cloning procedure follows clear and simple design rules and allows the assembly of multiple fragments with different sizes into one open reading frame. The final product can be directly integrated into the widely used Multisite Gateway(TM) cloning system, granting more flexibility when using a transgene in the context of multiple species. This adaptable and streamlined cloning procedure overcomes restrictions of “classical construct generation” and allows focusing on construct design.
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spelling pubmed-37921082013-10-10 Golden GATEway Cloning – A Combinatorial Approach to Generate Fusion and Recombination Constructs Kirchmaier, Stephan Lust, Katharina Wittbrodt, Joachim PLoS One Research Article The design and generation of DNA constructs is among the necessary but generally tedious tasks for molecular biologists and, typically, the cloning strategy is restricted by available restriction sites. However, increasingly sophisticated experiments require increasingly complex DNA constructs, with an intricacy that exceeds what is achievable using standard cloning procedures. Many transgenes such as inducible gene cassettes or recombination elements consist of multiple components that often require precise in-frame fusions. Here, we present an efficient protocol that facilitates the generation of these complex constructs. The golden GATEway cloning approach presented here combines two established cloning methods, namely golden Gate cloning and Multisite Gateway(TM) cloning. This allows efficient and seamless assembly as well as reuse of predefined DNA elements. The golden Gate cloning procedure follows clear and simple design rules and allows the assembly of multiple fragments with different sizes into one open reading frame. The final product can be directly integrated into the widely used Multisite Gateway(TM) cloning system, granting more flexibility when using a transgene in the context of multiple species. This adaptable and streamlined cloning procedure overcomes restrictions of “classical construct generation” and allows focusing on construct design. Public Library of Science 2013-10-07 /pmc/articles/PMC3792108/ /pubmed/24116091 http://dx.doi.org/10.1371/journal.pone.0076117 Text en © 2013 Kirchmaier et al http://creativecommons.org/licenses/by/4.0/ 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 properly credited.
spellingShingle Research Article
Kirchmaier, Stephan
Lust, Katharina
Wittbrodt, Joachim
Golden GATEway Cloning – A Combinatorial Approach to Generate Fusion and Recombination Constructs
title Golden GATEway Cloning – A Combinatorial Approach to Generate Fusion and Recombination Constructs
title_full Golden GATEway Cloning – A Combinatorial Approach to Generate Fusion and Recombination Constructs
title_fullStr Golden GATEway Cloning – A Combinatorial Approach to Generate Fusion and Recombination Constructs
title_full_unstemmed Golden GATEway Cloning – A Combinatorial Approach to Generate Fusion and Recombination Constructs
title_short Golden GATEway Cloning – A Combinatorial Approach to Generate Fusion and Recombination Constructs
title_sort golden gateway cloning – a combinatorial approach to generate fusion and recombination constructs
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3792108/
https://www.ncbi.nlm.nih.gov/pubmed/24116091
http://dx.doi.org/10.1371/journal.pone.0076117
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