Oligonucleotide-Directed Mutagenesis by Elimination of a Unique Restriction Site (USE Mutagenesis).

In this method, two oligonucleotide primers are hybridized to the same strand of a denatured double-stranded recombinant plasmid. One primer (the mutagenic primer) introduces the desired mutation into the target sequences, whereas the second primer carries a mutation that destroys a unique restriction site (called "unique site elimination," or USE) in the plasmid. The product of the first part of the method is a heteroduplex plasmid consisting of a wild-type parental strand and a new full-length strand that carries the desired mutation but no longer contains the unique restriction site. In the second phase of the method, the mixed population is incubated with the restriction enzyme that cleaves the unique site. The wild-type molecules are linearized, and the mutated plasmids are resistant to digestion. The mixture of circular heteroduplex DNA and linear wild-type DNA is then used to transform a strain of

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