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.