Abstract
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.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Wang HH, Isaacs FJ, Carr PA et al (2009) Programming cells by multiplex genome engineering and accelerated evolution. Nature 460:894–898
Carr PA, Church GM (2009) Genome engineering. Nat Biotechnol 27:1151–1162
Gibson DG, Benders GA, Andrews-Pfannkoch C et al (2008) Complete chemical synthesis, assembly, and cloning of a Mycoplasma genitalium genome. Science 319:1215–1220
Kobayashi H, Kaern M, Araki M et al (2004) Programmable cells: interfacing natural and engineered gene networks. Proc Natl Acad Sci U S A 101:8414–8419
Stemmer WPC, Crameri A, Ha KD et al (1995) Single-step assembly of a gene and entire plasmid from large numbers of oligodeoxyribonucleotides. Gene 164:49–53
Gibson DG (2009) Synthesis of DNA fragments in yeast by one-step assembly of overlapping oligonucleotides. Nucleic Acids Res 37:6984–6990
Kosuri S, Eroshenko N, LeProust EM et al (2010) Scalable gene synthesis by selective amplification of DNA pools from high-fidelity microchips. Nat Biotechnol 28:1295–1299
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 29:4744–4750
Quan JY, Saaem I, Tang N et al (2011) Parallel on-chip gene synthesis and application to optimization of protein expression. Nat Biotechnol 29:449–452
Tian JD, Gong H, Sheng NJ et al (2004) Accurate multiplex gene synthesis from programmable DNA microchips. Nature 432:1050–1054
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 32:5011–5018
Zhou X, Cai S, Hong A et al (2004) Microfluidic PicoArray synthesis of oligodeoxynucleotides and simultaneous assembling of multiple DNA sequences. Nucleic Acids Res 32:5409–5417
Kim C, Kaysen J, Richmond K et al (2006) Progress in gene assembly from a MAS-driven DNA microarray. Microelectron Eng 83:1613–1616
Cleary MA, Kilian K, Wang YQ et al (2004) Production of complex nucleic acid libraries using highly parallel in situ oligonucleotide synthesis. Nat Methods 1:241–248
Linshiz G, Ben Yehezkel T, Kaplan S et al (2008) Recursive construction of perfect DNA molecules from imperfect oligonucleotides. Mol Syst Biol 4:1–10
Wan W, Li L, Xu Q et al (2014) Error removal in microchip-synthesized DNA using immobilized MutS. Nucleic Acids Res 42, e102
Hoover DM, Lubkowski J (2002) DNAWorks: an automated method for designing oligonucleotides for PCR-based gene synthesis. Nucleic Acids Res 30, e43
Brown J, Brown T, Fox KR (2001) Affinity of mismatch-binding protein MutS for heteroduplexes containing different mismatches. Biochem J 354:627–633
Cho M, Chung S, Heo SD et al (2007) A simple fluorescent method for detecting mismatched DNAs using a MutS-fluorophore conjugate. Biosens Bioelectron 22:1376–1381
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 621:193–199
Acknowledgments
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.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2017 Springer Science+Business Media New York
About this protocol
Cite this protocol
Wan, W., Wang, D., Gao, X., Hong, J. (2017). Immobilized MutS-Mediated Error Removal of Microchip-Synthesized DNA. In: Hughes, R. (eds) Synthetic DNA. Methods in Molecular Biology, vol 1472. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6343-0_17
Download citation
DOI: https://doi.org/10.1007/978-1-4939-6343-0_17
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-6341-6
Online ISBN: 978-1-4939-6343-0
eBook Packages: Springer Protocols