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 f or gene disruption. In a two-step polymerase chain reaction (PCR), a f ragment, corresponding to the terminator and promoter regions separate d 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 select ion in Saccharomyces cerevisiae (TRP1, HIS3 and KanMX). The final plas mids are used directly for gene disruption after linearization by the enzyme (e.g. AscI) specific for the rare restriction site. This approa ch was used to disrupt three open reading frames identified during the sequencing of COS14-1 from chromosome XIV of S. cerevisiae.