Protocol

Rapid and Efficient Site-directed Mutagenesis by the Single-tube Megaprimer PCR Method

This protocol was adapted from Molecular Cloning, 3rd edition, by Joseph Sambrook and David W. Russell. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2001

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INTRODUCTION

The "megaprimer" method of site-directed mutagenesis introduced by Kammann et al. (1989) uses three oligonucleotide primers and two rounds of PCR. One of the oligonucleotides is mutagenic, the other two being forward and reverse primers that lie upstream and downstream from the binding site for the mutagenic oligonucleotide. The mutagenic primer and the nearer of the external primers are used in the first PCR to generate and amplify a mutated fragment of DNA. This amplified fragment - the megaprimer - is used in the second PCR in conjunction with the remaining external . . . [Full Text of this Article]

MATERIALS

METHOD