Protocol

Oligonucleotide-directed Mutagenesis by Elimination of a Unique Restriction Site (USE Mutagenesis)

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

Two oligonucleotide primers are hybridized to the same strand of a denatured double-stranded recombinant plasmid. One primer (the mutagenic primer) introduces the desired mutation into the target sequences, and the second primer carries a mutation that destroys a unique restriction site in the plasmid. Both primers are elongated in a reaction catalyzed by bacteriophage T4 or T7 DNA polymerase. Nicks in the strand of newly synthesized DNA are sealed with bacteriophage T4 DNA ligase. The product of the first part of the method is a heteroduplex plasmid consisting of a wild-type parental strand and a new full-length strand that carries . . . [Full Text of this Article]

MATERIALS

METHOD