title: Improved gap repair cloning in yeast: treatment of the gapped vector with Taq DNA polymerase avoids vector self-ligation.
abstract: Gap repair is a fast and efficient method for assembling recombinant DNA molecules in Saccharomyces cerevisiae. This method produces a circular DNA molecule by homologous recombination between two or more linear DNA fragments, one of which is typically a vector carrying replicative sequences and a selective marker. This technique avoids laborious and costly in vitro purification and ligation of DNA. The DNA repair machinery can also close and ligate the linear vector by mechanisms other than homologous recombination, resulting in an empty vector. The frequency of these unwanted events can be lowered by removing the 5'-phosphate groups using phosphatase, which is the standard method used for in vitro ligation. However, phosphatase treatment is less effective for gap repair cloning than for in vitro ligation, presumably due to the ability of the S. cerevisiae DNA repair machinery to efficiently repair the missing phosphate group to allow religation. We have developed a more efficient method to prevent vector religation, based on treatment of the vector fragment with Taq DNA polymerase and dATP. This procedure prevents vector recircularization almost completely, facilitating the screening for true recombinant clones.
authors.affiliation: Centro de Investigação em Ciências da Saúde (CICS), Instituto Superior de Ciências da Saúde-Norte/CESPU, 4585-116 Gandra, PRD, Portugal.
authors.forename: Daniela
authors.initials: D
authors.lastname: Bessa
authors.forename: Filipa
authors.initials: F
authors.lastname: Pereira
authors.forename: Roxana
authors.initials: R
authors.lastname: Moreira
authors.forename: Björn
authors.initials: B
authors.lastname: Johansson
authors.forename: Odília
authors.initials: O
authors.lastname: Queirós
mesh_terms: D003001:Cloning, Molecular / Q000379:methods*; D004260:DNA Repair / Q000235:genetics*; D004270:DNA, Circular / Q000235:genetics*; D004271:DNA, Fungal / Q000235:genetics; D004274:DNA, Recombinant / Q000235:genetics*; D005822:Genetic Vectors; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D019914:Taq Polymerase / Q000378:metabolism*; D025062:Yarrowia / Q000235:genetics
chemical_list: D004270:DNA, Circular; D004271:DNA, Fungal; D004274:DNA, Recombinant; D019914:Taq Polymerase