Protocol

Site-specific Mutagenesis by Overlap Extension

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

The first 100 words of the full text of this article appear below.

INTRODUCTION

Four primers and three PCRs are used to create a site-specific mutation by overlap extension. One pair of primers is used to amplify DNA that contains the mutation site together with upstream sequences. The second pair of primers is used in a separate PCR to amplify DNA that contains the mutation site together with downstream sequences. The mutation(s) of interest is located in the region of overlap and therefore in both amplified fragments. The overlapping fragments are mixed, denatured, and annealed to generate heteroduplexes that can be extended and, in a third PCR, amplified into a full-length DNA using two . . . [Full Text of this Article]

MATERIALS

METHOD