Site-directed mutagenesis by overlap extension using the polymerase chain reaction

Steffan N. Ho, Henry D. Hunt, Robert M. Horton, Jeffrey K. Pullen, Larry R Pease

Research output: Contribution to journalArticle

6471 Citations (Scopus)

Abstract

Overlap extension represents a new approach to genetic engineering. Complementary oligodeoxyribo-nucleotide (oligo) primers and the polymerase chain reaction are used to generate two DNA fragments having overlapping ends. These fragments are combined in a subsequent 'fusion' reaction in which the overlapping ends anneal, allowing the 3 ' overlap of each strand to serve as a primer for the 3' extension of the complementary strand. The resulting fusion product is amplified further by PCR. Specific alterations in the nucleotide (nt) sequence can be introduced by incorporating nucleotide changes into the overlapping oligo primers. Using this technique of site-directed mutagenesis, three variants of a mouse major histocompatibility complex class-I gene have been generated, cloned and analyzed. Screening of mutant clones revealed at least a 98% efficiency of mutagenesis. All clones sequenced contained the desired mutations, and a low frequency of random substitution estimated to occur at approx. 1 in 4000 nt was detected. This method represents a significant improvement over standard methods of site-directed mutagenesis because it is much faster, simpler and approaches 100% efficiency in the generation of mutant product.

Original languageEnglish (US)
Pages (from-to)51-59
Number of pages9
JournalGene
Volume77
Issue number1
DOIs
StatePublished - Apr 15 1989

Fingerprint

Site-Directed Mutagenesis
Nucleotides
Polymerase Chain Reaction
Clone Cells
MHC Class I Genes
Genetic Engineering
Major Histocompatibility Complex
Mutagenesis
Mutation
DNA

Keywords

  • Genetic engineering
  • major histocompatibility complex mutants
  • oligodeoxyribonucleotide primers
  • recombinant DNA
  • sequencing
  • Taq polymerase

ASJC Scopus subject areas

  • Genetics

Cite this

Site-directed mutagenesis by overlap extension using the polymerase chain reaction. / Ho, Steffan N.; Hunt, Henry D.; Horton, Robert M.; Pullen, Jeffrey K.; Pease, Larry R.

In: Gene, Vol. 77, No. 1, 15.04.1989, p. 51-59.

Research output: Contribution to journalArticle

Ho, Steffan N. ; Hunt, Henry D. ; Horton, Robert M. ; Pullen, Jeffrey K. ; Pease, Larry R. / Site-directed mutagenesis by overlap extension using the polymerase chain reaction. In: Gene. 1989 ; Vol. 77, No. 1. pp. 51-59.
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