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Gene splicing and mutagenesis by PCR-driven overlap extension


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.

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Figure 1: PCR-mediated overlap extension can create specific nucleotide mutations or generate chimeric gene products.
Figure 2: Mutagenic primers b and c share complementary sequence.
Figure 3: Glycine to arginine mutation at position 83 of murine MHC Kb is created by overlap extension mutagenesis.


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We thank Rudy Hanson for his technical contributions to this paper and Virginia Van Keulen and Michael Bell for helpful discussions.

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Correspondence to Larry R Pease.

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Mayo Clinic is assigned intellectual property rights related to the described technology that have been and continue to be licensed in a non-exclusive manner to a number of non-academic private interests. Mayo Clinic and I (author) have received royalty payments in excess of $10,000. K. Heckman has no competing financial interest.

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Heckman, K., Pease, L. Gene splicing and mutagenesis by PCR-driven overlap extension. Nat Protoc 2, 924–932 (2007).

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