title: PCR Duplication: A One-Step Cloning-Free Method to Generate Duplicated Chromosomal Loci and Interference-Free Expression Reporters in Yeast.
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.
authors.affiliation: Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-ZMBH Allianz, University of Heidelberg, 69120, Heidelberg, Germany.
authors.forename: Florian
authors.initials: F
authors.lastname: Huber
authors.affiliation: Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-ZMBH Allianz, University of Heidelberg, 69120, Heidelberg, Germany.
authors.forename: Matthias
authors.initials: M
authors.lastname: Meurer
authors.affiliation: Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-ZMBH Allianz, University of Heidelberg, 69120, Heidelberg, Germany.
authors.forename: Daria
authors.initials: D
authors.lastname: Bunina
authors.affiliation: Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-ZMBH Allianz, University of Heidelberg, 69120, Heidelberg, Germany.
authors.forename: Ilia
authors.initials: I
authors.lastname: Kats
authors.affiliation: Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-ZMBH Allianz, University of Heidelberg, 69120, Heidelberg, Germany.
authors.forename: Céline I
authors.initials: CI
authors.lastname: Maeder
authors.affiliation: Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-ZMBH Allianz, University of Heidelberg, 69120, Heidelberg, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Stefl
authors.affiliation: Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-ZMBH Allianz, University of Heidelberg, 69120, Heidelberg, Germany.
authors.forename: Cyril
authors.initials: C
authors.lastname: Mongis
authors.affiliation: Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-ZMBH Allianz, University of Heidelberg, 69120, Heidelberg, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Knop
mesh_terms: D050756:Cyclin-Dependent Kinase Inhibitor Proteins / Q000235:genetics*; D020440:Gene Duplication / Q000235:genetics*; D017930:Genes, Reporter / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D011401:Promoter Regions, Genetic / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D050436:Regulatory Elements, Transcriptional / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics*; D014404:Tubulin / Q000235:genetics*
chemical_list: D050756:Cyclin-Dependent Kinase Inhibitor Proteins; C081353:FAR1 protein, S cerevisiae; D012333:RNA, Messenger; D029701:Saccharomyces cerevisiae Proteins; C100764:TUB4 protein, S cerevisiae; D014404:Tubulin