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Nucleic Acids Research 2005 33(6):e55; doi:10.1093/nar/gni053
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Published online 30 March 2005

© The Author 2005. Published by Oxford University Press. All rights reserved
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Methods Online

Correcting errors in synthetic DNA through consensus shuffling

Brock F. Binkowski1, Kathryn E. Richmond2, James Kaysen2, Michael R. Sussman1 and Peter J. Belshaw1,3,*

1Department of Biochemistry, University of Wisconsin-Madison Madison, WI 53706, USA 2Center for Nanotechnology, University of Wisconsin-Madison Madison, WI 53706, USA 3Department of Chemistry, University of Wisconsin-Madison Madison, WI 53706, USA

*To whom correspondence should be addressed. Tel: +1 608 262 2996; Fax: +1 608 265 4534; Email: belshaw{at}chem.wisc.edu

Received October 25, 2004. Revised March 1, 2005. Accepted March 1, 2005.

Although efficient methods exist to assemble synthetic oligonucleotides into genes and genomes, these suffer from the presence of 1–3 random errors/kb of DNA. Here, we introduce a new method termed consensus shuffling and demonstrate its use to significantly reduce random errors in synthetic DNA. In this method, errors are revealed as mismatches by re-hybridization of the population. The DNA is fragmented, and mismatched fragments are removed upon binding to an immobilized mismatch binding protein (MutS). PCR assembly of the remaining fragments yields a new population of full-length sequences enriched for the consensus sequence of the input population. We show that two iterations of consensus shuffling improved a population of synthetic green fluorescent protein (GFPuv) clones from ~60 to >90% fluorescent, and decreased errors 3.5- to 4.3-fold to final values of ~1 error per 3500 bp. In addition, two iterations of consensus shuffling corrected a population of GFPuv clones where all members were non-functional, to a population where 82% of clones were fluorescent. Consensus shuffling should facilitate the rapid and accurate synthesis of long DNA sequences.


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