title: Enrichment of error-free synthetic DNA sequences by CEL I nuclease.
abstract: As the availability of DNA sequence information has grown, so has the need to replicate DNA sequences synthetically. Synthetically produced DNA sequences allow the researcher to exert greater control over model systems and allow for the combinatorial design and construction of novel metabolic and regulatory pathways, as well as optimized protein-coding sequences for biotechnological applications. This utility has made synthetically produced DNA a hallmark of the molecular biosciences and a mainstay of synthetic biology. However, synthetically produced DNA has a significant shortcoming in that it typically has an error rate that is orders of magnitude higher when compared to DNA sequences derived directly from a biological source. This relatively high error rate adds to the cost and labor necessary to obtain sequence-verified clones from synthetically produced DNA sequences. This unit describes a protocol to enrich error-free sequences from a population of error-rich DNA via treatment with CEL I (Surveyor) endonuclease. This method is a straightforward and quick way of reducing the error content of synthetic DNA pools and reliably reduces the error rates by >6-fold per round of treatment.
authors.affiliation: The University of Texas at Austin, Applied Research Laboratories, Department of Chemistry and Biochemistry, Center for Systems and Synthetic Biology, Austin, Texas, USA.
authors.forename: Randall A
authors.initials: RA
authors.lastname: Hughes
authors.forename: Aleksandr E
authors.initials: AE
authors.lastname: Miklos
authors.forename: Andrew D
authors.initials: AD
authors.lastname: Ellington
mesh_terms: D004247:DNA / Q000138:chemical synthesis*; D004587:Electrophoresis, Agar Gel; D004720:Endonucleases; D005813:Genes, Synthetic; D009154:Mutation; D058615:Synthetic Biology / Q000191:economics / Q000379:methods*
chemical_list: D004247:DNA; D004720:Endonucleases; C406922:CEL I nuclease