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. 1982 Sep 11;10(17):5161–5171. doi: 10.1093/nar/10.17.5161

Deletion loop mutagenesis: a novel method for the construction of point mutations using deletion mutants.

D Kalderon, B A Oostra, B K Ely, A E Smith
PMCID: PMC320862  PMID: 6292832

Abstract

Deletion loop mutagenesis is a new, general method for site-directed mutagenesis that allows point mutations to the introduced within a sequence of DNA defined by a previously isolated deletion mutant. Wild type and deletion mutant DNA are cloned into a bacterial plasmid and each is cleaved with a different single cut restriction enzyme. Heteroduplexes are formed between the two DNAs to produce circular molecules containing a nick in each strand and a single-stranded deletion loop. The deletion loops are mutagenised using sodium bisulphite and the DNA transfected directly into a uracil repair deficient strain of Escherichia coli. Up to half of the resultant clones contain DNA produced by replication of the wild-type length strand and bear mutations exclusively within the target area. An example is given in which a deletion mutant lacking 21 nucleotides from the region coding for SV40 large-T was used. Eight of the possible nine target cytosine residues were mutagenised. The method described is specific, efficient and simple.

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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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