Abstract
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.
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References
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 164:49–53
Tian J, Gong H, Sheng N, Zhou X, Gulari E, Gao X, Church G (2004) Accurate multiplex gene synthesis from programmable DNA microchips. Nature 432:1050–1054
Czar MJ, Anderson JC, Bader JS, Peccoud J (2009) Gene synthesis demystified. Trends Biotechnol 27:63–72
Tian J, Ma K, Saaem I (2009) Advancing high-throughput gene synthesis technology. Mol Biosyst 5:714–722
Hughes RA, Miklos AE, Ellington AD (2011) Gene synthesis: methods and applications. Methods Enzymol 498:277–309
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 35:1992–2002
Hoover DM, Lubkowski J (2002) DNAWorks: an automated method for designing oligonucleotides for PCR-based gene synthesis. Nucleic Acids Res 30:e43
Hoover D (2012) Using DNAWorks in designing oligonucleotides for PCR-based gene synthesis. Methods Mol Biol 852:215–223
Richardson SM, Nunley PW, Yarrington RM, Boeke JD, Bader JS (2010) GeneDesign 3.0 is an updated synthetic biology toolkit. Nucleic Acids Res 38:2603–2606
Acknowledgments
The authors are grateful to Peter Weigele for valuable suggestions. We also thank Laurie Mazzola for DNA sequencing and John Buswell for oligonucleotide synthesis. We are especially thankful for the environment and support provided by Don Comb.
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Vaisvila, R., Bitinaite, J. (2013). Gene Synthesis by Assembly of Deoxyuridine-Containing Oligonucleotides. In: Samuelson, J. (eds) Enzyme Engineering. Methods in Molecular Biology, vol 978. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-293-3_12
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DOI: https://doi.org/10.1007/978-1-62703-293-3_12
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Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-292-6
Online ISBN: 978-1-62703-293-3
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