Gene splicing and mutagenesis by PCR-driven overlap extension

Karin L. Heckman, Larry R Pease

Research output: Contribution to journalArticle

690 Citations (Scopus)

Abstract

Extension of overlapping gene segments by PCR is a simple, versatile technique for site-directed mutagenesis and gene splicing. Initial PCRs generate overlapping gene segments that are then used as template DNA for another PCR to create a full-length product. Internal primers generate overlapping, complementary 3′ ends on the intermediate segments and introduce nucleotide substitutions, insertions or deletions for site-directed mutagenesis, or for gene splicing, encode the nucleotides found at the junction of adjoining gene segments. Overlapping strands of these intermediate products hybridize at this 3′ region in a subsequent PCR and are extended to generate the full-length product amplified by flanking primers that can include restriction enzyme sites for inserting the product into an expression vector for cloning purposes. The highly efficient generation of mutant or chimeric genes by this method can easily be accomplished with standard laboratory reagents in approximately 1 week.

Original languageEnglish (US)
Pages (from-to)924-932
Number of pages9
JournalNature Protocols
Volume2
Issue number4
DOIs
StatePublished - Apr 2007

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Mutagenesis
Genes
Overlapping Genes
Polymerase Chain Reaction
Site-Directed Mutagenesis
Nucleotides
Genetic Vectors
DNA
Substitution reactions
Enzymes

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

Cite this

Gene splicing and mutagenesis by PCR-driven overlap extension. / Heckman, Karin L.; Pease, Larry R.

In: Nature Protocols, Vol. 2, No. 4, 04.2007, p. 924-932.

Research output: Contribution to journalArticle

Heckman, Karin L. ; Pease, Larry R. / Gene splicing and mutagenesis by PCR-driven overlap extension. In: Nature Protocols. 2007 ; Vol. 2, No. 4. pp. 924-932.
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