‘Shotgun DNA synthesis’ for the high-throughput construction of large DNA molecules

We developed a highly scalable ‘shotgun’ DNA synthesis technology by utilizing microchip oligonucleotides, shotgun assembly and next-generation sequencing technology. A pool of microchip oligonucleotides targeting a penicillin biosynthetic gene cluster were assembled into numerous random fragments, and tagged with 20 bp degenerate barcode primer pairs. An optimal set of error-free fragments were identified by high-throughput DNA sequencing, selectively amplified using the barcode sequences, and successfully assembled into the target gene cluster.

[1]  George M Church,et al.  High-fidelity gene synthesis by retrieval of sequence-verified DNA identified using high-throughput pyrosequencing. , 2010, Nature biotechnology.

[2]  S. Turner,et al.  Real-time DNA sequencing from single polymerase molecules. , 2010, Methods in enzymology.

[3]  Tuval Ben Yehezkel,et al.  Recursive construction of perfect DNA molecules from imperfect oligonucleotides , 2008, Molecular systems biology.

[4]  S. Turner,et al.  A flexible and efficient template format for circular consensus sequencing and SNP detection , 2010, Nucleic acids research.

[5]  Nicholas C Tang,et al.  Parallel on-chip gene synthesis and application to optimization of protein expression , 2011, Nature Biotechnology.

[6]  James R. Knight,et al.  Genome sequencing in microfabricated high-density picolitre reactors , 2005, Nature.

[7]  J. Martín,et al.  The cluster of penicillin biosynthetic genes. Identification and characterization of the pcbAB gene encoding the alpha-aminoadipyl-cysteinyl-valine synthetase and linkage to the pcbC and penDE genes. , 1990, The Journal of biological chemistry.

[8]  K. Sykes,et al.  High-quality gene assembly directly from unpurified mixtures of microarray-synthesized oligonucleotides , 2010, Nucleic acids research.

[9]  Duhee Bang,et al.  Gene synthesis by circular assembly amplification , 2008, Nature Methods.

[10]  J Craig Venter,et al.  Chemical synthesis of the mouse mitochondrial genome , 2010, Nature Methods.

[11]  G. Church,et al.  Accurate multiplex gene synthesis from programmable DNA microchips , 2004, Nature.

[12]  Jingdong Tian,et al.  Error correction in gene synthesis technology. , 2012, Trends in biotechnology.

[13]  Peter A Carr,et al.  Protein-mediated error correction for de novo DNA synthesis. , 2004, Nucleic acids research.

[14]  Peter A Carr,et al.  Genome engineering , 2009, Nature Biotechnology.

[15]  Santiago Garcia-Vallvé,et al.  Working toward a new NIOSH. , 1996, Nucleic Acids Res..

[16]  Sriram Kosuri,et al.  Scalable gene synthesis by selective amplification of DNA pools from high-fidelity microchips , 2010, Nature Biotechnology.

[17]  D. Bang,et al.  Hierarchical gene synthesis using DNA microchip oligonucleotides. , 2011, Journal of biotechnology.

[18]  D. Bang,et al.  A Fluorescence Selection Method for Accurate Large‐Gene Synthesis , 2010, Chembiochem : a European journal of chemical biology.

[19]  F. Sanger,et al.  Nucleotide sequence of bacteriophage lambda DNA. , 1982, Journal of molecular biology.

[20]  Thomas H Segall-Shapiro,et al.  Creation of a Bacterial Cell Controlled by a Chemically Synthesized Genome , 2010, Science.

[21]  Jingdong Tian,et al.  Error correction of microchip synthesized genes using Surveyor nuclease , 2011, Nucleic acids research.