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PCR Duplication: A One-Step Cloning-Free Method to Generate Duplicated Chromosomal Loci and Interference-Free Expression Reporters in Yeast
Here, we report on a novel PCR targeting-based strategy called ‘PCR duplication’ that enables targeted duplications of genomic regions in the yeast genome using a simple PCR-based approach. To demonstrate its application we first duplicated the promoter of the FAR1 gene in yeast and simultaneously i...
Autores principales: | , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2014
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4262419/ https://www.ncbi.nlm.nih.gov/pubmed/25493941 http://dx.doi.org/10.1371/journal.pone.0114590 |
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author | Huber, Florian Meurer, Matthias Bunina, Daria Kats, Ilia Maeder, Céline I. Štefl, Martin Mongis, Cyril Knop, Michael |
author_facet | Huber, Florian Meurer, Matthias Bunina, Daria Kats, Ilia Maeder, Céline I. Štefl, Martin Mongis, Cyril Knop, Michael |
author_sort | Huber, Florian |
collection | PubMed |
description | Here, we report on a novel PCR targeting-based strategy called ‘PCR duplication’ that enables targeted duplications of genomic regions in the yeast genome using a simple PCR-based approach. To demonstrate its application we first duplicated the promoter of the FAR1 gene in yeast and simultaneously inserted a GFP downstream of it. This created a reporter for promoter activity while leaving the FAR1 gene fully intact. In another experiment, we used PCR duplication to increase the dosage of a gene in a discrete manner, from 1× to 2x. Using TUB4, the gene encoding for the yeast γ-tubulin, we validated that this led to corresponding increases in the levels of mRNA and protein. PCR duplication is an easy one-step procedure that can be adapted in different ways to permit rapid, disturbance-free investigation of various genomic regulatory elements without the need for ex vivo cloning. |
format | Online Article Text |
id | pubmed-4262419 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2014 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-42624192014-12-15 PCR Duplication: A One-Step Cloning-Free Method to Generate Duplicated Chromosomal Loci and Interference-Free Expression Reporters in Yeast Huber, Florian Meurer, Matthias Bunina, Daria Kats, Ilia Maeder, Céline I. Štefl, Martin Mongis, Cyril Knop, Michael PLoS One Research Article Here, we report on a novel PCR targeting-based strategy called ‘PCR duplication’ that enables targeted duplications of genomic regions in the yeast genome using a simple PCR-based approach. To demonstrate its application we first duplicated the promoter of the FAR1 gene in yeast and simultaneously inserted a GFP downstream of it. This created a reporter for promoter activity while leaving the FAR1 gene fully intact. In another experiment, we used PCR duplication to increase the dosage of a gene in a discrete manner, from 1× to 2x. Using TUB4, the gene encoding for the yeast γ-tubulin, we validated that this led to corresponding increases in the levels of mRNA and protein. PCR duplication is an easy one-step procedure that can be adapted in different ways to permit rapid, disturbance-free investigation of various genomic regulatory elements without the need for ex vivo cloning. Public Library of Science 2014-12-10 /pmc/articles/PMC4262419/ /pubmed/25493941 http://dx.doi.org/10.1371/journal.pone.0114590 Text en © 2014 Huber 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 Huber, Florian Meurer, Matthias Bunina, Daria Kats, Ilia Maeder, Céline I. Štefl, Martin Mongis, Cyril Knop, Michael PCR Duplication: A One-Step Cloning-Free Method to Generate Duplicated Chromosomal Loci and Interference-Free Expression Reporters in Yeast |
title | PCR Duplication: A One-Step Cloning-Free Method to Generate Duplicated Chromosomal Loci and Interference-Free Expression Reporters in Yeast |
title_full | PCR Duplication: A One-Step Cloning-Free Method to Generate Duplicated Chromosomal Loci and Interference-Free Expression Reporters in Yeast |
title_fullStr | PCR Duplication: A One-Step Cloning-Free Method to Generate Duplicated Chromosomal Loci and Interference-Free Expression Reporters in Yeast |
title_full_unstemmed | PCR Duplication: A One-Step Cloning-Free Method to Generate Duplicated Chromosomal Loci and Interference-Free Expression Reporters in Yeast |
title_short | PCR Duplication: A One-Step Cloning-Free Method to Generate Duplicated Chromosomal Loci and Interference-Free Expression Reporters in Yeast |
title_sort | pcr duplication: a one-step cloning-free method to generate duplicated chromosomal loci and interference-free expression reporters in yeast |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4262419/ https://www.ncbi.nlm.nih.gov/pubmed/25493941 http://dx.doi.org/10.1371/journal.pone.0114590 |
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