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Updated: Jul 7, 2026

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli
Published on: March 16, 2011
Oligonucleotide-directed mutagenesis without phenotypic selection
1National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina, USA.
Abstract:
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.
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