Synthetic DNA pp 217-235 | Cite as

Immobilized MutS-Mediated Error Removal of Microchip-Synthesized DNA

  • Wen Wan
  • Dongmei Wang
  • Xiaolian Gao
  • Jiong HongEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1472)


Applications of microchip-synthesized oligonucleotides for de novo gene synthesis are limited primarily by their high error rates. The mismatch binding protein MutS, which can specifically recognize and bind to mismatches, is one of the cheapest tools for error correction of synthetic DNA. Here, we describe a protocol for removing errors in microchip-synthesized oligonucleotides and for the assembly of DNA segments using these oligonucleotides. This protocol can also be used in traditional de novo gene DNA synthesis.

Key words

MutS Microchip-synthesized oligonucleotides Gene assemble Error removal De novo gene synthesis 



This work was supported by a grant-in-aid from the National Natural Science Foundation of China (31270149), the National High Technology Research and Development Program (2012AA02A708), Anhui Provincial Natural Science Foundation (1608085MC47),the Fundamental Research Funds for the Central Universities (WK2070000059), the China Postdoctoral Science Foundation (2015M580546). This work also earned technical support from the Core Facility Center for Life Sciences, University of Science and Technology of China.


  1. 1.
    Wang HH, Isaacs FJ, Carr PA et al (2009) Programming cells by multiplex genome engineering and accelerated evolution. Nature 460:894–898CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Carr PA, Church GM (2009) Genome engineering. Nat Biotechnol 27:1151–1162CrossRefPubMedGoogle Scholar
  3. 3.
    Gibson DG, Benders GA, Andrews-Pfannkoch C et al (2008) Complete chemical synthesis, assembly, and cloning of a Mycoplasma genitalium genome. Science 319:1215–1220CrossRefPubMedGoogle Scholar
  4. 4.
    Kobayashi H, Kaern M, Araki M et al (2004) Programmable cells: interfacing natural and engineered gene networks. Proc Natl Acad Sci U S A 101:8414–8419CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Stemmer WPC, Crameri A, Ha KD et al (1995) Single-step assembly of a gene and entire plasmid from large numbers of oligodeoxyribonucleotides. Gene 164:49–53CrossRefPubMedGoogle Scholar
  6. 6.
    Gibson DG (2009) Synthesis of DNA fragments in yeast by one-step assembly of overlapping oligonucleotides. Nucleic Acids Res 37:6984–6990CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Kosuri S, Eroshenko N, LeProust EM et al (2010) Scalable gene synthesis by selective amplification of DNA pools from high-fidelity microchips. Nat Biotechnol 28:1295–1299CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Gao XL, LeProust E, Zhang H et al (2001) A flexible light-directed DNA chip synthesis gated by deprotection using solution photogenerated acids. Nucleic Acids Res 29:4744–4750CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Quan JY, Saaem I, Tang N et al (2011) Parallel on-chip gene synthesis and application to optimization of protein expression. Nat Biotechnol 29:449–452CrossRefPubMedGoogle Scholar
  10. 10.
    Tian JD, Gong H, Sheng NJ et al (2004) Accurate multiplex gene synthesis from programmable DNA microchips. Nature 432:1050–1054CrossRefPubMedGoogle Scholar
  11. 11.
    Richmond KE, Li MH, Rodesch MJ et al (2004) Amplification and assembly of chip-eluted DNA (AACED): a method for high-throughput gene synthesis. Nucleic Acids Res 32:5011–5018CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Zhou X, Cai S, Hong A et al (2004) Microfluidic PicoArray synthesis of oligodeoxynucleotides and simultaneous assembling of multiple DNA sequences. Nucleic Acids Res 32:5409–5417CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Kim C, Kaysen J, Richmond K et al (2006) Progress in gene assembly from a MAS-driven DNA microarray. Microelectron Eng 83:1613–1616CrossRefGoogle Scholar
  14. 14.
    Cleary MA, Kilian K, Wang YQ et al (2004) Production of complex nucleic acid libraries using highly parallel in situ oligonucleotide synthesis. Nat Methods 1:241–248CrossRefPubMedGoogle Scholar
  15. 15.
    Linshiz G, Ben Yehezkel T, Kaplan S et al (2008) Recursive construction of perfect DNA molecules from imperfect oligonucleotides. Mol Syst Biol 4:1–10CrossRefGoogle Scholar
  16. 16.
    Wan W, Li L, Xu Q et al (2014) Error removal in microchip-synthesized DNA using immobilized MutS. Nucleic Acids Res 42, e102CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Hoover DM, Lubkowski J (2002) DNAWorks: an automated method for designing oligonucleotides for PCR-based gene synthesis. Nucleic Acids Res 30, e43CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Brown J, Brown T, Fox KR (2001) Affinity of mismatch-binding protein MutS for heteroduplexes containing different mismatches. Biochem J 354:627–633CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Cho M, Chung S, Heo SD et al (2007) A simple fluorescent method for detecting mismatched DNAs using a MutS-fluorophore conjugate. Biosens Bioelectron 22:1376–1381CrossRefPubMedGoogle Scholar
  20. 20.
    Hong J, Ye X, Wang Y et al (2008) Bioseparation of recombinant cellulose-binding module-proteins by affinity adsorption on an ultra-high-capacity cellulosic adsorbent. Anal Chim Acta 621:193–199CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  • Wen Wan
    • 1
  • Dongmei Wang
    • 1
  • Xiaolian Gao
    • 1
    • 2
  • Jiong Hong
    • 1
    • 2
    Email author
  1. 1.School of Life ScienceUniversity of Science and Technology of ChinaHefeiPeople’s Republic of China
  2. 2.Hefei National Laboratory for Physical Science at the MicroscaleHefei, 230026People’s Republic of China

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