Directed Evolution Library Creation

Volume 1179 of the series Methods in Molecular Biology pp 189-206


Assembly of Designed Oligonucleotides: A Useful Tool in Synthetic Biology for Creating High-Quality Combinatorial DNA Libraries

  • Carlos G. Acevedo-RochaAffiliated withOrganische Synthese, Max-Planck-Institut für KohlenforschungPhilipps-Universität Marburg, Fachbereich Chemie
  • , Manfred T. ReetzAffiliated withOrganische Synthese, Max-Planck-Institut für KohlenforschungPhilipps-Universität Marburg, Fachbereich Chemie Email author 

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The method dubbed Assembly of Designed Oligonucleotides (ADO) is a powerful tool in synthetic biology to create combinatorial DNA libraries for gene, protein, metabolic, and genome engineering. In directed evolution of proteins, ADO benefits from using reduced amino acid alphabets for saturation mutagenesis and/or DNA shuffling, but all 20 canonical amino acids can be also used as building blocks. ADO is performed in a two-step reaction. The first involves a primer-free, polymerase cycling assembly or overlap extension PCR step using carefully designed overlapping oligonucleotides. The second step is a PCR amplification using the outer primers, resulting in a high-quality and bias-free double-stranded DNA library that can be assembled with other gene fragments and/or cloned into a suitable plasmid subsequently. The protocol can be performed in a few hours. In theory, neither the length of the DNA library nor the number of DNA changes has any limits. Furthermore, with the costs of synthetic DNA dropping every year, after an initial investment is made in the oligonucleotides, these can be exchanged for alternative ones with different sequences at any point in the process, fully exploiting the potential of creating highly diverse combinatorial libraries. In the example chosen here, we show the construction of a high-quality combinatorial ADO library targeting sixteen different codons simultaneously with nonredundant degenerate codons encoding various reduced alphabets of four amino acids along the heme region of the monooxygenase P450-BM3.

Key words

Synthetic biology Directed evolution Intellectual property DNA shuffling Synthetic shuffling Degenerate homoduplex recombination Gene assembly mutagenesis Saturation mutagenesis Combinatorial libraries Reduced amino acid alphabets