Site-Directed Mutagenesis of Whole Viral Genomes

This chapter introduces an efficient and accurate site-directed mutagenesis protocol, which allows the color selection of mutants through the simultaneous activation or deactivation of the α-peptide of β-galactosidase. It uses doublestranded plasmid DNA as the mutational template. This protocol can efficiently create mutations of large inserts at multiple sites simultaneously and can be used to perform multiple rounds of mutation on the same construct. Thus, constructs containing whole open-reading frames and whole viral genomes can be subjected to site-directed mutagenesis and used for subsequent functional studies.