Please cite as: CSH Protocols; 2006; doi:10.1101/pdb.prot3817

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Oligonucleotide-directed Mutagenesis of Single-stranded DNA

Joseph Sambrook and David W. Russell

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

Uracil-containing bacteriophage M13 DNA, generated in Preparation of Uracil-containing Single-stranded Bacteriophage M13 DNA, is used in the classic double-primer method of oligonucleotide-mediated mutagenesis of Zoller and Smith (1984, 1987) to generate a heteroduplex molecule with uracil in the template strand and thymine in the strand synthesized in the in vitro reaction. Transformation of this DNA into an ung+ strain results in destruction of the template strand, with consequent suppression of production of wild-type bacteriophages. Up to 80% of the plaques are therefore derived by replication of the uracil-free strand. Because synthesis of this strand has been primed by a . . . [Full Text of this Article]


MATERIALS


METHOD


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