Fusion PCR, a One-Step Variant of the “Megaprimer” Method of Mutagenesis

Fusion PCR, a one-step variant of the "megaprimer" method of 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...

Creating random mutagenesis libraries using megaprimer PCR of whole plasmid.

The conventional method for cloning a DNA fragment is to insert it into a vector and ligate it. Although this method is commonly used, it is labor intensive because the ratio and concentrations of the DNA insert and the vector need optimizing. Even then, the resultant library is often plagued with unwanted plasmids that have no inserts or multiple inserts. These species have to be eradicated to...

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...

Simplified one-tube "megaprimer" polymerase chain reaction mutagenesis.

Site-directed mutagenesis has become an essential tool in the investigation of gene structure and function as well as the products of gene expression, namely RNA and protein. Selected positions in a DNA sequence are changed, and the eVects of the mutations are studied in vitro and/or in vivo. Although many diVerent mutagenic strategies are available [1], the simplicity, cost eVectiveness, and a...