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)


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.


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.


  1. 1.
    Deng, W. P. and Nickoloff, J. A. (1992) Site-directed mutagenesis of virtually any plasmid by eliminating a unique site. Anal. Biochem. 200, 81–88.PubMedCrossRefGoogle Scholar
  2. 2.
    Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning A Laboratory Manual, 2nd ed, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.Google Scholar
  3. 3.
    Carter, P. (1987) Improved oligonucleotide-directed mutagenesis using M13 vectors. Methods Enzymol. 154, 382–403.PubMedCrossRefGoogle Scholar
  4. 4.
    Kunkel, T. A., Roberts, J. D., and Zakour, R. A. (1987) Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 154, 367–382.PubMedCrossRefGoogle Scholar
  5. 5.
    Kunkel, T. A., (1985) The use of phosphorothioate-modified DNA in restriction enzyme reactions to prepare nicked DNA. Proc. Natl. Acad. Sci. USA 82, 488–492.PubMedCrossRefGoogle Scholar
  6. 6.
    Lewis, M. K. and Thompson, D. V. (1990) Efficient site-directed in vitro mutagenesis using ampicillin selection. Nucleic Acids Res. 18, 3439–3443.PubMedCrossRefGoogle Scholar
  7. 7.
    Taylor, J. W., Ott, J., and Eckstein, F. (1985) The rapid generation of oligonucleotide-directed mutations at high frequency using phosphorothioate-modified DNA. Nucleic Acids Res. 13, 8764–8785.Google Scholar
  8. 8.
    Taylor, J. W., Schmidt, W., Cosstick, R., Okruszek, A., and Eckstein, F. (1985) The rapid generation of oligonucleotide-directed mutations at high frequency using phosphorothioate-modified DNA. Nucleic Acids Res. 13, 8779–8785.Google Scholar
  9. 9.
    Vandeyr, M., Weiner, M., Hutton, C., and Batt, C. (1988) A simple and rapid method for the selection of oligodeoxyribonucleotide-directed mutations. Gene 65, 129–133.CrossRefGoogle Scholar
  10. 10.
    Cohen, S. N., Chang, A. C. Y., and Hsu, L. (1972) Nonchromosomal antibiotic resistance in bacteria: genetic transformation of Escherichia coli by R-factor DNA. Proc. Natl. Acad. Sci. USA 69, 2110–2114.PubMedCrossRefGoogle Scholar
  11. 11.
    Conley, E. C. and Saunders, J. R. (1984) Recombination-dependent recircularization of linearized pBR322 plasmid DNA following transformation of Escherichia coli. Mol. Gen. Genet. 194, 211–218.PubMedCrossRefGoogle Scholar
  12. 12.
    Zhu, L. (1992) Highly efficient site-directed mutagenesis of dsDNA plasmids. CLONTECHniques VII(1), 1–5.Google Scholar
  13. 13.
    Zhu, L. and Chen, H. (1992) In vitro generation of multiple-site mutations and precise large deletions. CLONTECHniques VII(2), 9–11.Google Scholar
  14. 14.
    Van Aelst, L., Barr, M., Marcus, S., Polverino, A., and Wigler, M. (1993) Complex formation between RAS and RAF and other protein kinases. Proc. Natl. Acad. Sci. USA 90, 6213–6217.PubMedCrossRefGoogle Scholar
  15. 15.
    Haught, C., Wilkinson, D. L., Zgafas, K., and Harrison, R. G. (1994) A method to insert a DNA fragment into a double-stranded plasmid. BioTechniques 16, 46–48.PubMedGoogle Scholar
  16. 16.
    Zell, R. and Fritz, H (1987) DNA mismatch repair in Escherichia coli counteracting the hydrolytic deamination of 5-methyl-cytosine residues. EMBO J. 6, 1809–1815.PubMedGoogle Scholar
  17. 17.
    Chung, C. T., Niemela, S. L., and Miller, R. H. (1989) One-step preparation of competent Escherichia coli: transformation and storage of bacterial cells in the same solution. Proc. Natl. Acad. Sci. USA 86, 2172–2175.PubMedCrossRefGoogle Scholar
  18. 18.
    Protocol for Transformer™ Site-Directed Mutagenesis Kit (1994) Clontech #K1600-1, Palo Alto, CA.Google Scholar
  19. 19.
    Holmes, D. S. and Quigley, M. (1981) A rapid boiling method for the preparation of bacterial plasmids. Anal. Biochem. 114, 193–197.PubMedCrossRefGoogle Scholar
  20. 20.
    Masumune, Y. and Richardson, C. A. (1971) Strand displacement during deoxyri-bonucleic acid synthesis at single-strand breaks. J. Biol. Chem. 246, 2692–2701.Google Scholar
  21. 21.
    Nossal, N. G. (1974) DNA synthesis on a double-stranded DNA template by the T4 bacteriophage DNA polymerase and the T4 gene 32 DNA unwinding protein. J. Biol. Chem. 249, 5668–5676.PubMedGoogle Scholar
  22. 22.
    Perlak, F. J. (1990) Single-step, large-scale, site-directed in vitro mutagenesis using multiple oligonucleotides. Nucleic Acids Res. 18(24), 7457–7458.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 1996

Authors and Affiliations

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

Personalised recommendations