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Functional analysis reports. Precise gene disruption in Saccharomyces cerevisiae by double fusion polymerase chain reaction
Author(s) -
Amberg David C.,
Botstein David,
Beasley Ellen M.
Publication year - 1995
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/yea.320111307
Subject(s) - biology , saccharomyces cerevisiae , polymerase chain reaction , open reading frame , genetics , selectable marker , gene , yeast , computational biology , polymerase , fusion gene , sequence (biology) , saccharomyces , peptide sequence , plasmid
We adapted a fusion polymerase chain reaction (PCR) strategy to synthesize gene disruption alleles of any sequenced yeast gene of interest. The first step of the construction is to amplify sequences flanking the reading frame we want to disrupt and to amplify the selectable marker sequence. Then we fuse the upstream fragment to the marker sequence by fusion PCR, isolate this product and fuse it to the downstream sequence in a second fusion PCR reaction. The final PCR product can then be transformed directly into yeast. This method is rapid, relatively inexpensive, offers the freedom to choose from among a variety of selectable markers and allows one to construct precise disruptions of any sequenced open reading frame in Saccharomyces cerevisiae .