PCR- and ligation-mediated synthesis of marker cassettes with long flanking homology regions for gene disruption in Saccharomyces cerevisiae.

PCR-synthesis of marker cassettes with long flanking homology regions for gene disruptions in S. cerevisiae.

A PCR-method for fast production of disruption cassettes is introduced, that allows the addition of long flanking homology regions of several hundred base pairs (LFH-PCR) to a marker module. Such a disruption cassette was made by linking two PCR fragments produced from genomic DNA to kanMX6, a modification of dominant resistance marker making S. cerevisiae resistant to geneticin (G418). In a fi...

Disruption of six Saccharomyces cerevisiae ORFs on chromosome XII results in three lethal disruptants.

Six ORFs of unknown function from the left arm of chromosome XII of Saccharomyces cerevisiae were chosen for a reverse genetic approach to provide materials to assist in assignment of function. A two-step PCR using long-flanking homology was employed to amplify disruption cassettes consisting of a kanMX gene as selectable marker flanked by 250-350 bp long regions homologous to the target gene. ...

Rapid hypothesis testing with Candida albicans through gene disruption with short homology regions.

Disruption of newly identified genes in the pathogen Candida albicans is a vital step in determination of gene function. Several gene disruption methods described previously employ long regions of homology flanking a selectable marker. Here, we describe disruption of C. albicans genes with PCR products that have 50 to 60 bp of homology to a genomic sequence on each end of a selectable marker. W...

Isolation, Subtype Determination, Cloning and Expression of HBsAg Gene from an Iranian Carrier in Saccharomyces cerevisiae

The Hepatitis B Surface antigen ( HBsAg) gene was isolated from an Iranian HBeAg positive carrier by PCR. The gene was cloned in pUC19 for sequencing and pYES2 for expression in Saccharomyces cerevisiae, which pNF1 and pDF3 constructs were made respectively. The sequencing data showed that the isolated HBsAg gene shared more than 90% homology with the ayw subtype. The pDF3 was transferred into ...

Disruption of GAL1 Gene in Saccharomyces cerevisiae Leads to Higher Ethanol Production