A simple method for site-directed mutagenesis using the polymerase chain reaction

Anne Hemsley, Norman Arnheim, Michael Dennis Toney, Gino A Cortopassi, David J. Galas

Research output: Contribution to journalArticle

375 Citations (Scopus)

Abstract

We have developed a general and simple method for directing specific sequence changes in a plasmid using primed amplification by the polymerase chain reaction (PCR). The method is based on the amplification of the entire plasmid using primers that include the desired changes. The method is rapid, simple in its execution, and requires only minute amounts of plasmid template DNA. It is significant that there are no special requirements for appropriately placed restriction sites in the sequence to be manipulated. In our system the yield of transformants was high and the fraction of them harboring plasmids with only the desired change was consistently about 80%. The generality of the method should make it useful for the direct alteration of most cloned genes. The only limitation may be the total length of the plasmid to be manipulated. During the study we found that the Taq DNA polymerase used for PCR adds on a single extra base (usually an A) at the end of a large fraction of the newly synthesized chains. These had to be removed by the Klenow fragment of DNA polymerase to insure restoration of the gene sequence.

Original languageEnglish (US)
Pages (from-to)6545-6551
Number of pages7
JournalNucleic Acids Research
Volume17
Issue number16
DOIs
StatePublished - Aug 25 1989
Externally publishedYes

Fingerprint

Site-directed mutagenesis
Mutagenesis
Polymerase Chain Reaction
Polymerase chain reaction
Site-Directed Mutagenesis
Plasmids
DNA
Amplification
Genes
Gene
Restoration
Taq Polymerase
DNA Polymerase I
Template
DNA-Directed DNA Polymerase
Fragment
Entire
Restriction
Requirements

ASJC Scopus subject areas

  • Statistics, Probability and Uncertainty
  • Applied Mathematics
  • Health, Toxicology and Mutagenesis
  • Toxicology
  • Genetics(clinical)
  • Genetics

Cite this

A simple method for site-directed mutagenesis using the polymerase chain reaction. / Hemsley, Anne; Arnheim, Norman; Toney, Michael Dennis; Cortopassi, Gino A; Galas, David J.

In: Nucleic Acids Research, Vol. 17, No. 16, 25.08.1989, p. 6545-6551.

Research output: Contribution to journalArticle

Hemsley, Anne ; Arnheim, Norman ; Toney, Michael Dennis ; Cortopassi, Gino A ; Galas, David J. / A simple method for site-directed mutagenesis using the polymerase chain reaction. In: Nucleic Acids Research. 1989 ; Vol. 17, No. 16. pp. 6545-6551.
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