title: T7 Endonuclease I Mediates Error Correction in Artificial Gene Synthesis.
abstract: Efficacy of de novo gene synthesis largely depends on the quality of overlapping oligonucleotides used as template for PCR assembly. The error rate associated with current gene synthesis protocols limits the efficient and accurate production of synthetic genes, both in the small and large scales. Here, we analysed the ability of different endonuclease enzymes, which specifically recognize and cleave DNA mismatches resulting from incorrect impairments between DNA strands, to remove mutations accumulated in synthetic genes. The gfp gene, which encodes the green fluorescent protein, was artificially synthesized using an integrated protocol including an enzymatic mismatch cleavage step (EMC) following gene assembly. Functional and sequence analysis of resulting artificial genes revealed that number of deletions, insertions and substitutions was strongly reduced when T7 endonuclease I was used for mutation removal. This method diminished mutation frequency by eightfold relative to gene synthesis not incorporating an error correction step. Overall, EMC using T7 endonuclease I improved the population of error-free synthetic genes, resulting in an error frequency of 0.43 errors per 1 kb. Taken together, data presented here reveal that incorporation of a mutation-removal step including T7 endonuclease I can effectively improve the fidelity of artificial gene synthesis.
authors.affiliation: Centro Interdisciplinar de Investigação em Sanidade Animal (CIISA), Faculdade de Medicina Veterinária, Universidade de Lisboa, Avenida da Universidade Técnica, 1300-477, Lisbon, Portugal.|NZYTech Genes & Enzymes, Campus do Lumiar, Estrada do Paço do Lumiar, 1649-038, Lisbon, Portugal.
authors.forename: Ana Filipa
authors.initials: AF
authors.lastname: Sequeira
authors.affiliation: NZYTech Genes & Enzymes, Campus do Lumiar, Estrada do Paço do Lumiar, 1649-038, Lisbon, Portugal.
authors.forename: Catarina I P D
authors.initials: CI
authors.lastname: Guerreiro
authors.affiliation: Unité Mixte de Recherche (UMR) 7257, Centre National de la Recherche Scientifique (CNRS), Aix-Marseille Université, Architecture et Fonction des Macromolécules Biologiques (AFMB), Campus de Luminy, 163 Avenue de Luminy, 13288, Marseille Cedex 09, France.
authors.forename: Renaud
authors.initials: R
authors.lastname: Vincentelli
authors.affiliation: Centro Interdisciplinar de Investigação em Sanidade Animal (CIISA), Faculdade de Medicina Veterinária, Universidade de Lisboa, Avenida da Universidade Técnica, 1300-477, Lisbon, Portugal. cafontes@fmv.ulisboa.pt.|NZYTech Genes & Enzymes, Campus do Lumiar, Estrada do Paço do Lumiar, 1649-038, Lisbon, Portugal. cafontes@fmv.ulisboa.pt.
authors.forename: Carlos M G A
authors.initials: CM
authors.lastname: Fontes
mesh_terms: D020137:Base Pair Mismatch; D004247:DNA / Q000032:analysis / Q000592:standards*; D003850:Deoxyribonuclease I / Q000378:metabolism*; D005813:Genes, Synthetic; D049452:Green Fluorescent Proteins / Q000235:genetics; D009154:Mutation
chemical_list: D049452:Green Fluorescent Proteins; D004247:DNA; D003850:Deoxyribonuclease I
keywords: Enzyme mismatch cleavage (EMC); Error removal; Gene synthesis; T7 endonuclease I