Oligonucleotide-Directed Double-Strand Break Repair in Plasmids of Escherichia coli: A Method for Site-Specific Mutagenesis

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 defi...

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Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 1986-10, Vol.83 (19), p.7177-7181
Main Author: Mandecki, Wlodek
Format: Article
Language:English
Subjects:
Base Sequence
Biological and medical sciences
Chromosome Mapping
DNA
DNA Repair
Enzymes
Escherichia coli - genetics
Fundamental and applied biological sciences. Psychology
Genetic Engineering - methods
Genetic mutation
Molecular and cellular biology
Molecular genetics
Mutagenesis
Mutagenesis. Repair
Mutation
Nucleic Acid Hybridization
Nucleotides
Oligodeoxyribonucleotides - genetics
Oligonucleotides
Plasmids
Point mutation
Sequencing
Site directed mutagenesis
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Description
Summary: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, usign 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.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.83.19.7177