Correcting errors in synthetic DNA through consensus shuffling.

Brock F Binkowski; Kathryn E Richmond; James Kaysen; Michael R Sussman; Peter J Belshaw

doi:10.1093/nar/gni053

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PubMed · 15800206

Correcting errors in synthetic DNA through consensus shuffling.

Brock F Binkowski·Kathryn E Richmond·James Kaysen·Michael R Sussman·Peter J Belshaw

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Abstract

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 approximately 60 to >90% fluorescent, and decreased errors 3.5- to 4.3-fold to final values of approximately 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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Adenosine Triphosphatases Bacterial Proteins Base Sequence Consensus Sequence DNA Shuffling DNA-Binding Proteins Fluorescent Dyes Genes, Synthetic Green Fluorescent Proteins Models, Theoretical MutS DNA Mismatch-Binding Protein Mutation Oligodeoxyribonucleotides Polymerase Chain Reaction Evaluation Study Journal Article Research Support, U.S. Gov't, Non-P.H.S.Research Support, U.S. Gov't, P.H.S.

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Brock F Binkowski, Kathryn E Richmond, James Kaysen, Michael R Sussman, Peter J Belshaw. 2005-03-30. Correcting errors in synthetic DNA through consensus shuffling.. https://doi.org/10.1093/nar%2Fgni053

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