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.
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