PCR Duplication: A One-Step Cloning-Free Method to Generate Duplicated Chromosomal Loci and Interference-Free Expression Reporters in Yeast: e114590

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 simult...

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Veröffentlicht in:PloS one 2014-12, Vol.9 (12)
Hauptverfasser: Huber, Florian, Meurer, Matthias, Bunina, Daria, Kats, Ilia, Maeder, Celine I, Stefl, Martin, Mongis, Cyril, Knop, Michael
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Sprache:eng
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Zusammenfassung: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 gamma -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.
ISSN:1932-6203
DOI:10.1371/journal.pone.0114590