Oligonucleotide-directed mutagenesis without phenotypic selection.
A DNA sequence can be specifically altered by synthesizing the desired sequence change within an oligonucleotide, and then converting this into a biologically active circular DNA strand by using the oligonucleotide to prime in vitro synthesis on a single-stranded circular template. This protocol uses a DNA template containing a small number of uracil residues in place of thymine. Use of the uracil-containing template allows rapid and efficient recovery of mutants; in principle this same template can be applied to most of the other mutagenesis protocols in use.
Kunkel
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National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina, USA.
D005821:Genetic Techniques; D016254:Mutagenesis, Insertional; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D017354:Point Mutation; D012641:Selection, Genetic; D017384:Sequence Deletion