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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1986 Oct;83(19):7177–7181. doi: 10.1073/pnas.83.19.7177

Oligonucleotide-directed double-strand break repair in plasmids of Escherichia coli: a method for site-specific mutagenesis.

W Mandecki
PMCID: PMC386678  PMID: 3532104

Abstract

A DNA double-strand break can be efficiently repaired in Escherichia coli if an oligodeoxyribonucleotide is provided to direct the repair. The oligonucleotide must be at least 20 residues long and have a sequence identical to sequences flanking the break. The phenomenon can be used to introduce defined mutations into DNA in the area of a double-strand break. To obtain mutants, the oligonucleotide that carries a mutation and the denatured linearized plasmid DNA are introduced into E. coli by transformation. No enzymatic manipulation in vitro is required. The mutants can constitute up to 98% of the total number of transformants obtained. The efficiency of mutagenesis decreases as the distance between the mutation and the plasmid cleavage site increases. The universality of the method was tested by introducing mutations into four genes, using four plasmids and three E. coli strains, as well as eight restriction enzymes to linearize DNA. Several models of the oligonucleotide-directed DNA double-strand break repair are discussed.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

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