Sticky-end polymerase chain reaction method for systematic gene disruption in Saccharomyces cerevisiae.
We describe a new procedure for the generation of plasmids containing a large promoter and terminator region of a gene of interest, useful for gene disruption. In a two-step polymerase chain reaction (PCR), a fragment, corresponding to the terminator and promoter regions separated by a 16 bp sequence containing a rare restriction site (e.g. AscI), is synthesized (T-P fragment). This PCR fragment is cloned in vectors presenting a rare blunt-end cloning site and a yeast marker for selection in Saccharomyces cerevisiae (TRP1, HIS3 and KanMX). The final plasmids are used directly for gene disruption after linearization by the enzyme (e.g. AscI) specific for the rare restriction site. This approach was used to disrupt three open reading frames identified during the sequencing of COS14-1 from chromosome XIV of S. cerevisiae.
Maftahi
M
M
Institut National Agronomique Paris-Grignon, Laboratoire de Génétique Moléculaire et Cellulaire, INRA CNRS, Thiverval-Grignon, France.
Gaillardin
C
C
Nicaud
J M
JM
D019747:Aldose-Ketose Isomerases; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D004271:DNA, Fungal / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics; D005800:Genes, Fungal; D005819:Genetic Markers; D005822:Genetic Vectors; D006836:Hydro-Lyases / Q000235:genetics; D007613:Kanamycin Resistance / Q000235:genetics; D008957:Models, Genetic; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016133:Polymerase Chain Reaction / Q000379:methods*; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D012641:Selection, Genetic; D014170:Transformation, Genetic
D004271:DNA, Fungal; D005656:Fungal Proteins; D005819:Genetic Markers; D029701:Saccharomyces cerevisiae Proteins; C078382:endodeoxyribonuclease AscI; D015252:Deoxyribonucleases, Type II Site-Specific; D006836:Hydro-Lyases; C021434:imidazoleglycerolphosphate dehydratase; D019747:Aldose-Ketose Isomerases; C490353:TRP1 protein, S cerevisiae