Asymmetric overlap extension PCR method bypassing intermediate purification and the amplification of wild-type template in site-directed mutagenesis

Abstract

By combining asymmetric PCR and overlap extension, we developed a novel asymmetric overlap extension PCR (AOE-PCR) method for site-directed mutagenesis which bypassed the need for intermediate purification and excluded the amplification of a wild-type template. This method was used to introduce single base mutations into a small GTPase gene from cotton and to simultaneously introduce two mutations just by repeating this method using the first round AOE-PCR products as template. Our results suggested that the AOE-PCR method represents a valuable improvement of the original overlap extension PCR for site-directed mutagenesis.

Molecular Characterization of Zebrafish Oatp1d1 (Slco1d1), a Novel Organic Anion-transporting Polypeptide

“…Site-directed Mutagenesis-Site-directed mutagenesis was performed with the asymmetric overlap extension method as described previously (19).…”

An Improved Site-Directed Mutagenesis Procedure and Application for Identification of Mutant Clones

“…in traditional site-directed mutagenesis, two mutant fragments are amplified and purified separately in the first round of PCR and then are annealed and extended as templates in the second round (5). there are two reports of improved site-directed mutagenesis in which asymmetric PcR was used with oe-PcR without an intermediate purification step (21,23). In our study, we digested the methylated plasmid DnA with Dpni and then mixed the two mutant fragments for use as the template in the second round of amplification.…”

Are type IV pili involved in <i>Vibrio anguillarum</i> virulence towards sea bass (<i>Dicentrarchus labrax</i> L.) larvae?

“…Using AOE-PCR [41], an in-frame deletion of 378 bp of the pilA gene was constructed ( Figure 3). First, it was verified that the growth in MB was not affected in the mutant strain (data not shown).…”