Gene Synthesis

Volume 852 of the series Methods in Molecular Biology pp 11-21


Oligonucleotide Assembly in Yeast to Produce Synthetic DNA Fragments

  • Daniel G. GibsonAffiliated withDepartment of Synthetic Biology, J. Craig Venter Institute, Inc. Email author 

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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.

Key words

In vivo DNA assembly Yeast transformation Gene synthesis Oligonucleotides Synthetic biology