Recombination-mediated PCR-directed plasmid construction in vivo in yeast.
We have extended the technique of PCR-directed recombination in Saccharomyces cerevisiae to develop a simple method for plasmid or gene construction in the absence of suitable restriction sites. The DNA to be cloned is PCR-amplified with 30-40 bp of homology to a linearized yeast plasmid. Co-transformation into yeast results in homologous recombination at a position directed by the PCR oligonucleotides.
Oldenburg
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KR
Affymax Research Institute, 4001 Miranda Avenue, Palo Alto, CA 94304, USA.
Vo
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KT
Michaelis
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Paddon
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D003001:Cloning, Molecular / Q000379:methods; D017931:DNA Primers; D004274:DNA, Recombinant / Q000235:genetics; D005800:Genes, Fungal; D017930:Genes, Reporter / Q000235:genetics; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000235:genetics / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011995:Recombination, Genetic / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D014170:Transformation, Genetic / Q000235:genetics
D017931:DNA Primers; D004274:DNA, Recombinant; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins