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Sticky‐end polymerase chain reaction method for systematic gene disruption in Saccharomyces cerevisiae
Author(s) -
Maftahi M.,
Gaillardin C.,
Nicaud J.M.
Publication year - 1996
Publication title -
yeast
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.923
H-Index - 102
eISSN - 1097-0061
pISSN - 0749-503X
DOI - 10.1002/(sici)1097-0061(199607)12:9<859::aid-yea978>3.0.co;2-q
Subject(s) - biology , terminator (solar) , saccharomyces cerevisiae , plasmid , genetics , gene , restriction site , restriction enzyme , polymerase chain reaction , microbiology and biotechnology , restriction map , open reading frame , peptide sequence , ionosphere , physics , astronomy
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. Asc I), 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. Asc I) 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 .