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  • FAQ: What is the oligonucleotide-directed mutagenesis without phenotypic selection method?

    A single-stranded plasmid (e.g. M13) containing the DNA of interest is grown in the presence of uracil in an E. coli host tolerant to a U for T substitution. A primer containing a base change mutation is used to copy the plasmid. T7 DNA Polymerase then extends the primer all the way around the plasmid. Since the T7 DNA Polymerase does not displace DNA it stops at one copy. The plasmid is then transfected into an E. coli strain that degrades DNA containing uracil. The remaining strand contains the mutation.