Site-Directed Mutagenesis Using the Megaprimer Method

Zhidong Xu, Alessia Colosimo, and Dieter C. Gruenert

1. Introduction

Site-directed mutagenesis (SDM) is used to introduce a defined mutation into tar­get DNA of known sequence to study, for example, gene expression or protein struc­ture-function relationship. A number of polymerase chain reaction (PCR)-based mutagenesis methods have been developed (7). Among them, the "megaprimer" method is probably the simplest and most flexible (2-5). The megaprimer method utilizes two external oligonucleotide primers and one internal mutagenic primer in two rounds of PCR with a DNA template that contains the sequence to be mutated. The first round of PCR is carried out using one of the external primers and the mutagenic primer containing the desired mutation. This amplifies an intermediate PCR product that is purified and used as a "megaprimer" for the second round of PCR, along with the other external primer. The final PCR product is cloned into appropriate vectors and used in downstream applications.

Different modifications of the megaprimer method for SDM have been reported (2,3,6,7), each of which aims to increase the efficiency of mutagenesis. For example, the use of an excess of the purified "megaprimer" over the template DNA in the sec­ond round of PCR increases the frequency of mutant clones. Typically, these modifi­cations result in an overall efficiency of mutagenesis greater than 50%.

This part describes a simplified version of the megaprimer method based on a dilution of the products from the first-round PCR and their use as a template for the second-round amplification (see Fig. 1) (8). In the first PCR amplification, a wild-type sequence is mutated with a forward primer (A) and a mutagenic internal primer (M). The desired mutation(s) give rise to a new restriction enzyme cleavage site and/ or a functional mutation in the amplified product. A diluted aliquot of the first-round PCR reaction is used in a second round of PCR with external forward and reverse primers (A and B, respectively). During the first cycle, the mutated fragment is

Fig. 1. Schematic illustration of the megaprimer method for site-directed mutagenesis. The first-round PCR (PCR1) is performed using external forward primer (A) and mutagenic reverse primer (M) to amplify a mutagenic "

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