Intergenic Flip Flop, a method for systematic gene disruption and cloning in yeast.
We have developed a strategy named Intergenic Flip Flop which, for each gene, allows us to produce in one experiment both a disrupting cassette and a plasmid for gap repair. The same method can also be used to insert a reporter gene downstream from the promoter. This approach extends the polymerase chain reaction (PCR)-based strategy proposed by Maftahi et al. 1996. Our method consists of PCR amplification of the two flanking intergenic regions of the open reading frame (ORF) of interest, using two sets of oligonucleotides. Each PCR product is flanked by two short defined nucleotidic sequences with a unique restriction site, allowing subsequent hybridization between them. The association of the two amplimers by the complementary sequences either in the same orientation as in genomic DNA or in the opposite orientation, allows the generation, after PCR, of two distinct cassettes which can be cloned into suitable vectors. When the amplimer in the head-to-tail orientation is cloned in a vector containing a selective marker for yeast such as G418 resistance, it provides a disrupting cassette after cleavage at the unique restriction site introduced by the PCR between the two intergenic amplimers. The amplimer with a direct orientation cloned into a yeast vector, after cleavage at the unique restriction site between the intergenic regions, permits cloning by gap repair of the gene of interest in yeast. Finally, a reporter gene can be inserted in the same plasmid. We report here the successful application of this strategy to an ORF of chromosome XIV of Saccharomyces cerevisiae: N1216.
Mallet
L
L
Institut de Génétique et Microbiologie, URA1354 du CNRS, Université Paris-Sud, Orsay, France.
Jacquet
M
M
D003001:Cloning, Molecular / Q000379:methods*; D017931:DNA Primers; D004260:DNA Repair / Q000235:genetics*; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000235:genetics; D016681:Genome, Fungal; D016254:Mutagenesis, Insertional / Q000379:methods*; D009693:Nucleic Acid Hybridization; D016366:Open Reading Frames; D016133:Polymerase Chain Reaction / Q000379:methods*; D011401:Promoter Regions, Genetic / Q000235:genetics; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D014170:Transformation, Genetic
D017931:DNA Primers