Comments on a Recently Published PCR Method for Site-Directed Mutagenesis

Comments on a recently published PCR method for site-directed mutagenesis.

I would like to outline the similarities between a polymerase chain reaction (PCR)-based site-directed mutagenesis procedure published by Xu et al. in BioTechniques (3) and a similar procedure published by me and co-workers a few years ago in Nucleic Acids Research (1). Both the procedures have common experimental steps, such as performing an inverse PCR on a PCR-generated DNA fragment in which...

Combined Overlap Extension PCR Method for Improved Site Directed Mutagenesis

The combined overlap extension PCR (COE-PCR) method developed in this work combines the strengths of the overlap extension PCR (OE-PCR) method with the speed and ease of the asymmetrical overlap extension (AOE-PCR) method. This combined method allows up to 6 base pairs to be mutated at a time and requires a total of 40-45 PCR cycles. A total of eight mutagenesis experiments were successfully ca...

Modification of a PCR-based site-directed mutagenesis method.

Site-directed mutagenesis is a powerful tool for producing mutants to assess the importance of specific amino acid residues in a protein’s structure and/or function. We wanted to generate mutants of human ETS1 cDNA in the pET15b vector (Novagen, Madison, WI, USA) from which we had been producing wild-type protein for structural studies (11). Since we were subject to the constraints of this vect...

Rapid PCR method for site-directed mutagenesis on double-stranded plasmid DNA.

Construction of a recombinant vector for site-directed mutagenesis in Salmonella typhimurium

BACKGROUND: Among all common techniques in sitedirectedmutagenesis, λ Red recombinase system has beenwidely used to knock out chromosomal genes in bacteria. In thismethod, there is always the risk of DNA Linear digestion byhost's restriction enzymes that leads to the low frequency ofrecombination. OBJECTIVES:To overcome this, we constructeda recombinant vector to disrupt phoP gene in Salmonella...