A Simple Method to Introduce Internal Deletions or Mutations into Any Position of a Target DNA Sequence

Site-directed mutagenesis is a powerful tool used in modern molecular biology for protein and genetic engineering. A number of simple and elegant protocols are available to introduce mutations into a target DNA sequence. However, there are only a few methods described for deletion mutagenesis (1 –4 ). In this chapter, we demonstrate a simple method for creating internal deletions into any desired position in a target DNA sequence. The procedure involves replacement of the DNA region to be deleted with a restriction site for an endonuclease. This also facilitates the insertion of other DNA sequences into the site or if desired restoration of the deleted fragment. Introduction of the new restriction site also provides a useful diagnostic tool for screening for positive clones following the transformation procedure.