Simple Version of “Megaprimer” PCR for Site-Directed Mutagenesis

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...

Site-directed mutagenesis by double polymerase chain reaction : megaprimer method.

The "megaprimer" method (1) based on polymerase chain reaction (PCR) is one of the simplest and most versatile procedures of site-specific in vitro mutagenesis available to date. The method utilizes three oligonucleotide primers and two rounds of PCR performed on a DNA template containing the cloned gene that is to be mutated. The rationale of the method is shown schematically in Fig. 1 where A...

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.

"Megaprimer" method of PCR-based mutagenesis: the concentration of megaprimer is a critical factor.

The technique of site-directed mutagenesis (SDM) is widely used in the field of molecular biology to introduce mutations into DNA (5). The development of polymerase chain reaction (PCR)-based SDM has eliminated the need to clone DNA, therefore simplifying the technique and enabling the desired results to be achieved more quickly (3). The “megaprimer” method of PCR-based SDM incorporates three p...