A method for sequence-specific deletion mutagenesis.
We describe a novel procedure for the construction of deletion mutants. Existing exonuclease-based protocols are efficient at producing randomly positioned deletions over large regions of DNA, but are of limited use in targetted mutagenesis due to their inherent sequence-specificity. We have taken advantage of the Exonuclease III-resistant nature of alpha-thio-dNTPs, incorporated into the target DNA template by a primer extension reaction, to generate base-specific alpha-thio-dNTP terminated products. Following removal of the 5' overhanging strands, the products can be cloned to generate a nested set of deletions with single base-pair increments. We demonstrate the utility of this technique by isolating multiple deletions over a 40bp region of the human beta-interferon promoter.
King
P
P
Gene Expression Laboratory, Imperial Cancer Research Fund, London, UK.
Goodbourn
S
S
D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D003854:Deoxyribonucleotides / Q000235:genetics*; D006801:Humans; D016899:Interferon-beta / Q000235:genetics*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D013873:Thionucleotides / Q000235:genetics*
D003854:Deoxyribonucleotides; D013873:Thionucleotides; D016899:Interferon-beta