Gene Synthesis by Assembly of Deoxyuridine-Containing Oligonucleotides

Part of the Methods in Molecular Biology book series (MIMB, volume 978)


Gene synthesis is an invaluable technique in synthetic and molecular biology for synthesis of artificial genes, operons, and even genomes. In many cases the traditional methods for obtaining functional DNA sequences through cloning are not applicable due to the novelty of genetic material. Here, we describe the simple and economical DNA synthesis method based on USER™ technology. The method consists of (1) synthesis of building blocks up to 500 bp; (2) assembly of genes up to 3 kb; (3) error correction reassembly; and (4) assembly of operons up to 15 kb if needed.

Key words

Gene synthesis Polymerase chain reaction Uracil excision USER cloning Synthetic biology 


  1. 1.
    Stemmer WP, Crameri A, Ha KD, Brennan TM, Heyneker HL (1995) Single-step assembly of a gene and entire plasmid from large numbers of oligodeoxyribonucleotides. Gene 164:49–53PubMedCrossRefGoogle Scholar
  2. 2.
    Tian J, Gong H, Sheng N, Zhou X, Gulari E, Gao X, Church G (2004) Accurate multiplex gene synthesis from programmable DNA microchips. Nature 432:1050–1054PubMedCrossRefGoogle Scholar
  3. 3.
    Czar MJ, Anderson JC, Bader JS, Peccoud J (2009) Gene synthesis demystified. Trends Biotechnol 27:63–72PubMedCrossRefGoogle Scholar
  4. 4.
    Tian J, Ma K, Saaem I (2009) Advancing high-throughput gene synthesis technology. Mol Biosyst 5:714–722PubMedCrossRefGoogle Scholar
  5. 5.
    Hughes RA, Miklos AE, Ellington AD (2011) Gene synthesis: methods and applications. Methods Enzymol 498:277–309PubMedCrossRefGoogle Scholar
  6. 6.
    Bitinaite J, Rubino M, Varma KH, Schildkraut I, Vaisvila R, Vaiskunaite R (2007) USER friendly DNA engineering and cloning method by uracil excision. Nucleic Acids Res 35:1992–2002PubMedCrossRefGoogle Scholar
  7. 7.
    Hoover DM, Lubkowski J (2002) DNAWorks: an automated method for designing oligonucleotides for PCR-based gene synthesis. Nucleic Acids Res 30:e43PubMedCrossRefGoogle Scholar
  8. 8.
    Hoover D (2012) Using DNAWorks in designing oligonucleotides for PCR-based gene synthesis. Methods Mol Biol 852:215–223PubMedCrossRefGoogle Scholar
  9. 9.
    Richardson SM, Nunley PW, Yarrington RM, Boeke JD, Bader JS (2010) GeneDesign 3.0 is an updated synthetic biology toolkit. Nucleic Acids Res 38:2603–2606PubMedCrossRefGoogle Scholar

Copyright information

© Springer New York 2013

Authors and Affiliations

  1. 1.New England Biolabs, Inc.IpswichUSA

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