2001
DOI: 10.1002/yea.806
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Marker‐fusion PCR for one‐step mutagenesis of essential genes in yeast

Abstract: We describe a one-step gene replacement method based on fusion PCR that can be used to mutagenize essential genes at their endogenous locus. Marker-fusion PCR can facilitate transfer of alleles between strains as well as PCR-based techniques, such as site-directed and error-prone PCR mutagenesis, all without cloning or strain constructions. With this method, PCR is used to fuse a mutagenized fragment to an overlapping fragment containing a selectable marker flanked by regions of homology to the target. By tran… Show more

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Cited by 23 publications
(22 citation statements)
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“…A thermostable kanamycin resistance gene, HTK, was also amplified by PCR from pUC18/HTK plasmid DNA (30). The three DNA fragments were fused by the fusion PCR method (31) to obtain the pT7Blue/ttmutS2::HTK plasmid for ttmutS2 gene disruption. pT7Blue/ndx8::Hyg (hygromycin resistance gene) was a generous gift from Drs.…”
Section: Methodsmentioning
confidence: 99%
“…A thermostable kanamycin resistance gene, HTK, was also amplified by PCR from pUC18/HTK plasmid DNA (30). The three DNA fragments were fused by the fusion PCR method (31) to obtain the pT7Blue/ttmutS2::HTK plasmid for ttmutS2 gene disruption. pT7Blue/ndx8::Hyg (hygromycin resistance gene) was a generous gift from Drs.…”
Section: Methodsmentioning
confidence: 99%
“…The URA3 gene was directed to the CAN1 gene by first amplifying CAN1 using the primers bam-CAN1-for (CGGGATCCCAAAAGAAGACGC CGACATAGA) and bam-CAN1-rev (CGGGATCCCTACAA CATTCCAAAATTTGTCCC), cloning into the BamHI site of pRS406, cutting with BglII, and transforming RMY195. Secondly, the ADE2-TG(1-3) cassette was inserted into the NPR2 locus using fusion PCR (Kitazono et al 2002) and the primers 5ЈNPR2 1a (AACTCCACAACAA CACCCTCCAC) and 5ЈNPR2 1b (CGAAGTTTTGATGAGCCACACAGCGGGGCACATCTT CTAATGAGACAC) for the 5Ј end homology with NPR2; 3ЈNPR2 2a (CAGTAACAACGTTGGATCCGACCCCTCAAAA CTCCTCCTTAAATTCCTC) and 3ЈNPR2 2b (AATACCTT CTTCTTCGTCGCTG) for the 3Ј end homology with NPR2; and XhoIpsd158 rev (GGGTCGGATCCAACGTTGTTACTG) for the fusion of the 5Ј NPR2 homology with the ADE2-TG(1-3) from strain UCC5913. The process for creating RMY232 was the same as for RMY231 except UCC5913 was the parent strain.…”
Section: Yeast Strains and Plasmidsmentioning
confidence: 99%
“…Isolated H2B-mRFP PCR product including a 35S terminator was fused to the indicated AtFH5 promoter regions and 5ЈUTR by marker-fusion PCR (Kitazono et al, 2002) then recombined with Alligator2 (Bensmihen et al, 2004) by Gateway cloning (Invitrogen, Carlsbad, CA, USA). Alligator2 was first digested with HindIII and EcoRV to remove the 2ϫ35S promoter, ATG and 3ϫHA tag.…”
Section: Construction Of Vectorsmentioning
confidence: 99%