Site-Directed Mutagenesis Using Oligonucleotide-Based Recombineering

Methods enabling mutational analysis of distinct chromosomal locations, like site-directed mutagenesis, insertion of foreign sequences or in-frame deletions, have become of fast growing interest since complete bacterial genome sequences became available. Various approaches have been described to modify any nucleotide(s) in almost any manner. Some genetic engineering technologies do not rely on the in vitro reactions carried out by restriction enzymes and DNA ligases (Sawitzke et al., 2001). Complicated genetic constructs that seem to be impossible to generate in vitro can be created within one week using in vivo technologies (Sawitzke et al., 2001).