Oligonucleotide assembly in yeast to produce synthetic DNA fragments.
The yeast Saccharomyces cerevisiae can take up and assemble at least 38 overlapping single-stranded oligonucleotides and a linear double-stranded vector in one transformation event. These oligonucleotides can overlap by as few as 20 bp and can be as long as 200 nucleotides in length to produce kilobase-sized synthetic DNA molecules. A protocol for designing the oligonucleotides to be assembled, transforming them into yeast, and confirming their assembly is described here. This straightforward scheme for assembling chemically synthesized oligonucleotides can be a useful tool for building synthetic DNA molecules.
Department of Synthetic Biology, J. Craig Venter Institute, Inc., Rockville, MD, USA. dgibson@jcvi.org
Daniel G
DG
Gibson
D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D014170:Transformation, Genetic
D009838:Oligodeoxyribonucleotides; D004247:DNA