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In Vitro Site-Directed Mutagenesis Using the Unique Restriction Site Elimination (USE) Method

Part of the Methods In Molecular Medicine™ book series (MIMB, volume 57)

Abstract

In vitro site-directed mutagenesis has been widely used in vector modification, and in gene and protein structure/function studies (1,2). This procedure typically employs one or more oligonuleotides to introduce defined mutations into a DNA target of known sequence (2, 3, 4, 5, 6, 7, 8, 9). A variation of this procedure, termed the USE (Unique Restriction Site Elimination) mutagenesis method (1), offers two important—and unique—advantages: specific base changes can be introduced into virtually any double-stranded plasmid; and plasmids carrying the desired mutation can be highly enriched by selecting against the parental (wild-type) plasmid. The USE strategy employs two oligonucleotide primers: one primer (the mutagenic primer) produces the desired mutation, whereas the second primer (the selection primer) mutates a restriction site unique to the plasmid for the purpose of selection.

Keywords

Restriction Enzyme Digestion Mutagenic Primer Selection Primer Mutant Plasmid Tn10 Insertion 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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Copyright information

© Humana Press Inc. 1996

Authors and Affiliations

  • Li Zhu
    • 1
  1. 1.Clontech LaboratoriesPalo Alto

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