Send to

Choose Destination
Yeast. 1996 Jul;12(9):859-68.

Sticky-end polymerase chain reaction method for systematic gene disruption in Saccharomyces cerevisiae.

Author information

Institut National Agronomique Paris-Grignon, Laboratoire de Génétique Moléculaire et Cellulaire, INRA CNRS, Thiverval-Grignon, France.


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.

[Indexed for MEDLINE]
Free full text

Supplemental Content

Full text links

Icon for Wiley
Loading ...
Support Center