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Molecular cloning using polymerase chain reaction, an educational guide for cellular engineering

BACKGROUND: Over the last decades, molecular cloning has transformed biological sciences. Having profoundly impacted various areas such as basic science, clinical, pharmaceutical, and environmental fields, the use of recombinant DNA has successfully started to enter the field of cellular engineering...

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Autores principales: Hoseini, Sayed Shahabuddin, Sauer, Martin G
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2015
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4350901/
https://www.ncbi.nlm.nih.gov/pubmed/25745516
http://dx.doi.org/10.1186/1754-1611-9-2
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author Hoseini, Sayed Shahabuddin
Sauer, Martin G
author_facet Hoseini, Sayed Shahabuddin
Sauer, Martin G
author_sort Hoseini, Sayed Shahabuddin
collection PubMed
description BACKGROUND: Over the last decades, molecular cloning has transformed biological sciences. Having profoundly impacted various areas such as basic science, clinical, pharmaceutical, and environmental fields, the use of recombinant DNA has successfully started to enter the field of cellular engineering. Here, the polymerase chain reaction (PCR) represents one of the most essential tools. Due to the emergence of novel and efficient PCR reagents, cloning kits, and software, there is a need for a concise and comprehensive protocol that explains all steps of PCR cloning starting from the primer design, performing PCR, sequencing PCR products, analysis of the sequencing data, and finally the assessment of gene expression. It is the aim of this methodology paper to provide a comprehensive protocol with a viable example for applying PCR in gene cloning. RESULTS: Exemplarily the sequence of the tdTomato fluorescent gene was amplified with PCR primers wherein proper restriction enzyme sites were embedded. Practical criteria for the selection of restriction enzymes and the design of PCR primers are explained. Efficient cloning of PCR products into a plasmid for sequencing and free web-based software for the consecutive analysis of sequencing data is introduced. Finally, confirmation of successful cloning is explained using a fluorescent gene of interest and murine target cells. CONCLUSIONS: Using a practical example, comprehensive PCR-based protocol with important tips was introduced. This methodology paper can serve as a roadmap for researchers who want to quickly exploit the power of PCR-cloning but have their main focus on functional in vitro and in vivo aspects of cellular engineering. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/1754-1611-9-2) contains supplementary material, which is available to authorized users.
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spelling pubmed-43509012015-03-06 Molecular cloning using polymerase chain reaction, an educational guide for cellular engineering Hoseini, Sayed Shahabuddin Sauer, Martin G J Biol Eng Methodology BACKGROUND: Over the last decades, molecular cloning has transformed biological sciences. Having profoundly impacted various areas such as basic science, clinical, pharmaceutical, and environmental fields, the use of recombinant DNA has successfully started to enter the field of cellular engineering. Here, the polymerase chain reaction (PCR) represents one of the most essential tools. Due to the emergence of novel and efficient PCR reagents, cloning kits, and software, there is a need for a concise and comprehensive protocol that explains all steps of PCR cloning starting from the primer design, performing PCR, sequencing PCR products, analysis of the sequencing data, and finally the assessment of gene expression. It is the aim of this methodology paper to provide a comprehensive protocol with a viable example for applying PCR in gene cloning. RESULTS: Exemplarily the sequence of the tdTomato fluorescent gene was amplified with PCR primers wherein proper restriction enzyme sites were embedded. Practical criteria for the selection of restriction enzymes and the design of PCR primers are explained. Efficient cloning of PCR products into a plasmid for sequencing and free web-based software for the consecutive analysis of sequencing data is introduced. Finally, confirmation of successful cloning is explained using a fluorescent gene of interest and murine target cells. CONCLUSIONS: Using a practical example, comprehensive PCR-based protocol with important tips was introduced. This methodology paper can serve as a roadmap for researchers who want to quickly exploit the power of PCR-cloning but have their main focus on functional in vitro and in vivo aspects of cellular engineering. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/1754-1611-9-2) contains supplementary material, which is available to authorized users. BioMed Central 2015-01-19 /pmc/articles/PMC4350901/ /pubmed/25745516 http://dx.doi.org/10.1186/1754-1611-9-2 Text en © Hoseini and Sauer; licensee BioMed Central. 2015 This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
spellingShingle Methodology
Hoseini, Sayed Shahabuddin
Sauer, Martin G
Molecular cloning using polymerase chain reaction, an educational guide for cellular engineering
title Molecular cloning using polymerase chain reaction, an educational guide for cellular engineering
title_full Molecular cloning using polymerase chain reaction, an educational guide for cellular engineering
title_fullStr Molecular cloning using polymerase chain reaction, an educational guide for cellular engineering
title_full_unstemmed Molecular cloning using polymerase chain reaction, an educational guide for cellular engineering
title_short Molecular cloning using polymerase chain reaction, an educational guide for cellular engineering
title_sort molecular cloning using polymerase chain reaction, an educational guide for cellular engineering
topic Methodology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4350901/
https://www.ncbi.nlm.nih.gov/pubmed/25745516
http://dx.doi.org/10.1186/1754-1611-9-2
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