
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.
Adenosine Triphosphatases, Base Sequence, Green Fluorescent Proteins, DNA Shuffling, Models, Theoretical, Polymerase Chain Reaction, MutS DNA Mismatch-Binding Protein, DNA-Binding Proteins, Bacterial Proteins, Oligodeoxyribonucleotides, Consensus Sequence, Mutation, Genes, Synthetic, Genetics, Methods Online, Fluorescent Dyes
Adenosine Triphosphatases, Base Sequence, Green Fluorescent Proteins, DNA Shuffling, Models, Theoretical, Polymerase Chain Reaction, MutS DNA Mismatch-Binding Protein, DNA-Binding Proteins, Bacterial Proteins, Oligodeoxyribonucleotides, Consensus Sequence, Mutation, Genes, Synthetic, Genetics, Methods Online, Fluorescent Dyes
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