n efficient method to perform gene disruption plasmid by overlap extension PCR for site directed mutagenesis in Brucella abortus is demonstrated in this study. This method is based on three standard PCR and one fusion PCR reactions for construction of gene disruption construct and then insertion into a vector for performing of gene disruption plasmid. The present method in despite of conventional methods, is not restriction site dependent and require only one step host cell cloning.
