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Kunkel mutagenesis (dUTP system mutagenesis)

A method to construct a site-specific mutated gene and to select it away from the wild-type. A strain of E. coli with only weak dUTPase activity has high levels of dUTP and consequently incorporates dUTP into a plasmid DNA in competition with dTTP. This plasmid is used in an in vitro system as a template for DNA synthesis with a primer into which the desired mutation has been incorporated. The duplex, containing the wild-type sequence with Us replacing Ts (U-DNA) complementary to a strand with the usual DNA bases and incorporating the mutation, is introduced into a wild-type bacterium that can remove the inappropriate bases from, and thus inactivate, the U-DNA strand and leave the mutated strand to be replicated.


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