Molecular Cloning Fourth Edition, A Laboratory Manual, by Michael R. Green and Joseph Sambrook

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

(Protocol summary only for purposes of this preview site)

In this method, 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, whereas the second primer carries a mutation that destroys a unique restriction site (called unique site elimination, or USE) in the plasmid (see Fig. 1). 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 the desired mutation but no longer contains the unique restriction site. The population of plasmids therefore consists of (1) wild-type molecules that, for one reason or another, were never used as templates for DNA synthesis primed by the two oligonucleotide primers and (2) heteroduplex molecules that have lost the unique restriction site and gained the desired mutation.


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(Limited time special offer.) Molecular Cloning Fourth Edition, A Laboratory Manual, by Michael R. Green and Joseph Sambrook
Molecular Cloning Fourth Edition, A Laboratory Manual, by Michael R. Green and Joseph Sambrook

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