High-quality gene assembly directly from unpurified mixtures of microarray-synthesized oligonucleotides

To meet the growing demand for synthetic genes more robust, scalable and inexpensive gene assembly technologies must be developed. Here, we present a protocol for high-quality gene assembly directly from low-cost marginal-quality microarray-synthesized oligonucleotides. Significantly, we eliminated the time- and money-consuming oligonucleotide purification steps through the use of hybridization-based selection embedded in the assembly process. The protocol was tested on mixtures of up to 2000 oligonucleotides eluted directly from microarrays obtained from three different chip manufacturers. These mixtures containing <5% perfect oligos, and were used directly for assembly of 27 test genes of different sizes. Gene quality was assessed by sequencing, and their activity was tested in coupled in vitro transcription/translation reactions. Genes assembled from the microarray-eluted material using the new protocol matched the quality of the genes assembled from >95% pure column-synthesized oligonucleotides by the standard protocol. Both averaged only 2.7 errors/kb, and genes assembled from microarray-eluted material without clonal selection produced only 30% less protein than sequence-confirmed clones. This report represents the first demonstration of cost-efficient gene assembly from microarray-synthesized oligonucleotides. The overall cost of assembly by this method approaches 5¢ per base, making gene synthesis more affordable than traditional cloning.

[1]  E. Wimmer,et al.  Putting Synthesis into Biology: A Viral View of Genetic Engineering through De Novo Gene and Genome Synthesis , 2009, Chemistry & Biology.

[2]  C. Dieffenbach,et al.  PCR primer: a laboratory manual. , 2003 .

[3]  S. Ho,et al.  Site-directed mutagenesis by overlap extension using the polymerase chain reaction. , 1989, Gene.

[4]  J Craig Venter,et al.  Generating a synthetic genome by whole genome assembly: φX174 bacteriophage from synthetic oligonucleotides , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[5]  Timothy B. Stockwell,et al.  Complete Chemical Synthesis, Assembly, and Cloning of a Mycoplasma genitalium Genome , 2008, Science.

[6]  Jean Peccoud,et al.  Gene synthesis demystified. , 2009, Trends in biotechnology.

[7]  Kimberly A. Smith,et al.  POSaM: a fast, flexible, open-source, inkjet oligonucleotide synthesizer and microarrayer , 2004, Genome Biology.

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

[9]  C. Dieffenbach,et al.  Setting up a PCR laboratory. , 1993, PCR methods and applications.

[10]  S. Lo,et al.  Gene synthesis by a LCR-based approach: high-level production of leptin-L54 using synthetic gene in Escherichia coli. , 1998, Biochemical and biophysical research communications.

[11]  Onnop Srivannavit,et al.  Microfluidic PicoArray synthesis of oligodeoxynucleotides and simultaneous assembling of multiple DNA sequences. , 2004, Nucleic acids research.

[12]  Sarah J Kodumal,et al.  Total synthesis of long DNA sequences: synthesis of a contiguous 32-kb polyketide synthase gene cluster. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[13]  D. Endy,et al.  Refactoring bacteriophage T7 , 2005, Molecular systems biology.

[14]  W. Stemmer,et al.  Single-step assembly of a gene and entire plasmid from large numbers of oligodeoxyribonucleotides. , 1995, Gene.

[15]  S. Hoffman,et al.  Codon Optimization of Gene Fragments EncodingPlasmodium falciparum Merzoite Proteins Enhances DNA Vaccine Protein Expression and Immunogenicity in Mice , 2001, Infection and Immunity.

[16]  J. Shaffer,et al.  Hybridization of synthetic oligodeoxyribonucleotides to ΦX 174 DNA: the effect of single base pair mismatch , 1979 .

[17]  A. Paul,et al.  Chemical Synthesis of Poliovirus cDNA: Generation of Infectious Virus in the Absence of Natural Template , 2002, Science.

[18]  O. Zelphati,et al.  Transcriptionally Active Polymerase Chain Reaction (TAP) , 2002, The Journal of Biological Chemistry.

[19]  P. Sharp,et al.  The codon Adaptation Index--a measure of directional synonymous codon usage bias, and its potential applications. , 1987, Nucleic acids research.

[20]  Richard H. Lathrop,et al.  Computationally Optimised DNA Assembly of synthetic genes , 2008, Int. J. Bioinform. Res. Appl..

[21]  Franco Cerrina,et al.  Amplification and assembly of chip-eluted DNA (AACED): a method for high-throughput gene synthesis. , 2004, Nucleic acids research.

[22]  G. Wahl,et al.  Hybridization of nucleic acids immobilized on solid supports. , 1984, Analytical biochemistry.

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

[24]  Changhan Kim,et al.  Progress in gene assembly from a MAS-driven DNA microarray , 2006 .

[25]  Malek Faham,et al.  A comprehensive assay for targeted multiplex amplification of human DNA sequences , 2008, Proceedings of the National Academy of Sciences.

[26]  J. Newcomb.  The new age of biological engineering: Implications for the US economy , 2007 .

[27]  K. Itakura,et al.  Hybridization of synthetic oligodeoxyribonucleotides to phi chi 174 DNA: the effect of single base pair mismatch. , 1979, Nucleic acids research.

[28]  K. Wood,et al.  Firefly Luciferase Engineered for Improved Genetic Reporting , 1994 .

[29]  E. Southern,et al.  Optimised ligation of oligonucleotides by thermal ligases: comparison of Thermus scotoductus and Rhodothermus marinus DNA ligases to other thermophilic ligases. , 2000, Nucleic acids research.

[30]  P. Sharp,et al.  Predicting gene expression level from codon usage bias. , 2007, Molecular biology and evolution.

[31]  Edwin M. Southern,et al.  Electrochemically directed synthesis of oligonucleotides for DNA microarray fabrication , 2005, Nucleic acids research.

[32]  Feng Gao,et al.  Non‐Polymerase‐Cycling‐Assembly‐Based Chemical Gene Synthesis: Strategies, Methods, and Progress , 2008 .

[33]  Kuosheng Ma,et al.  Advancing high-throughput gene synthesis technology. , 2009, Molecular bioSystems.