Site-Directed Mutagenesis Using a Uracil-Containing Phagemid Template

Abstract

The ability to introduce specific changes into almost any given DNA sequence has revolutionized the analysis of cloned genes. This technique has enabled researchers to identify regions necessary for the regulation of gene expression. Also, it was and still is instrumental to learn about the importance of functional domains or even single amino acids of proteins. Of the many useful methods available for site-directed mutagenesis, in this chapter I describe a protocol that is based on the “Kunkel Method” (1,2). This method allows the generation of point mutations, deletions, and insertions for a given DNA sequence with high efficiency. I have used this procedure successfully many times and most recently to map protein interaction sites within the activation domain of the transcription factor E2F (3).