title: Correcting errors in synthetic DNA through consensus shuffling.
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.
authors.affiliation: Department of Biochemistry, University of Wisconsin-Madison Madison, WI 53706, USA.
authors.forename: Brock F
authors.initials: BF
authors.lastname: Binkowski
authors.forename: Kathryn E
authors.initials: KE
authors.lastname: Richmond
authors.forename: James
authors.initials: J
authors.lastname: Kaysen
authors.forename: Michael R
authors.initials: MR
authors.lastname: Sussman
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Belshaw
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism; D001483:Base Sequence; D016384:Consensus Sequence; D032621:DNA Shuffling / Q000379:methods*; D004268:DNA-Binding Proteins / Q000378:metabolism; D005456:Fluorescent Dyes; D005813:Genes, Synthetic; D049452:Green Fluorescent Proteins / Q000235:genetics; D008962:Models, Theoretical; D051719:MutS DNA Mismatch-Binding Protein; D009154:Mutation; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism; D016133:Polymerase Chain Reaction
chemical_list: D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D005456:Fluorescent Dyes; D009838:Oligodeoxyribonucleotides; D049452:Green Fluorescent Proteins; D000251:Adenosine Triphosphatases; D051719:MutS DNA Mismatch-Binding Protein