A Novel Method for Site-directed Mutagenesis and Its Application in DdsA Mutant Research

To develop the gene mutagenesis technique, a novel method of site-directed mutagenesis was introduced. The half-blunt end restriction enzyme sites were used in this new method. Comparing with the traditional overlapping extension mutagenesis, this method could replace any nucleotides covered the gene sequence. Meanwhile, the mutant gene was cloned in the T-vector directly that make sequencing convenient. Nineteen mutants of DdsA (decaprenyl diphosphate synthase), focused on four residues, were generated via this method. The results showed that the new gene mutagenesis method was efficient.