| 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/GCC-, GCC-, GGC- or GGC-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/CC- or GG-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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