title: Effect of base-mismatch in the cohesive ends of oligonucleotide in gene cloning.
abstract: The tolerance of mismatched nucleotides between the cohesive ends of insert and target DNAs in gene cloning has been investigated. An oligonucleotide duplex with a cohesive end GGCC-5' or variation was ligated to the 5'-CCGG end of a linearized plasmid. The ligation mixture was used in the transformation of E. coli. A single-base mismatch, such as 5'-CCGG/AGCC-, GACC-, GGAC- or GGCA-5' (mismatch underlined), was well tolerated in the cloning of the oligonucleotide duplex, with efficiency lower than the fully complementary ends. Double-base mismatch 5'-CCGG/AACC- or GGAA-5' resulted in further decrease of cloning efficiency. Via a similar approach, a tetracycline resistance gene was successfully inserted into a pUC-type plasmid.
authors.affiliation: Institute for Biological Sciences, National Research Council of Canada, Ottawa, ON.
authors.forename: W L
authors.initials: WL
authors.lastname: Sung
authors.forename: D M
authors.initials: DM
authors.lastname: Zahab
authors.forename: C K
authors.initials: CK
authors.lastname: Luk
authors.forename: B S
authors.initials: BS
authors.lastname: Chan
mesh_terms: D001482:Base Composition; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D013753:Tetracycline Resistance / Q000235:genetics
chemical_list: D009838:Oligodeoxyribonucleotides