Directed mutagenesis using PCR

Site-directed mutagenesis using PCR-mediated introduction of silent mutations.

The alteration of a specific sequence of DNA using site-directed mutagenesis (SDM) provides an effective tool to probe the structure and function of gene products. With the advent of polymerase chain reaction (PCR), several new techniques were developed that facilitate SDM. An oligonucleotide containing mismatches at the desired site of mutation is used to generate a population of mutant fragme...

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...

PCR-generated cross-over linkers for site-directed mutagenesis.

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...

Simple version of "megaprimer" PCR for site-directed mutagenesis.

use in restriction-enzyme analysis. This procedure is cost-effective because it saves the use of expensive miniprep kits for use with only positively identified recombinant clones and limits typically time-consuming miniprep steps. Our method can be accomplished in a few minutes in two microcentrifuge tubes with minimal enzyme, expense, no phenol/chloroform extractions and no ethanol precipitat...