Effect of base-mismatch in the cohesive ends of oligonucleotide in gene cloning.

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.