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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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