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PCR duplication: a one-step cloning-free method to generate duplicated chromosomal loci and interference-free expression reporters in yeast

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Item Type:Article
Title:PCR duplication: a one-step cloning-free method to generate duplicated chromosomal loci and interference-free expression reporters in yeast
Creators Name:Huber, F., Meurer, M., Bunina, D., Kats, I., Maeder, C.I., Stefl, M., Mongis, C. and Knop, M.
Abstract: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.
Keywords:Cyclin-Dependent Kinase Inhibitor Proteins, Gene Duplication, Genetic Promoter Regions, Messenger RNA, Polymerase Chain Reaction, Reporter Genes, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Transcriptional Regulatory Elements, Tubulin
Source:PLoS ONE
ISSN:1932-6203
Publisher:Public Library of Science
Volume:9
Number:12
Page Range:e114590
Date:10 December 2014
Official Publication:https://doi.org/10.1371/journal.pone.0114590
PubMed:View item in PubMed

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