Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension

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

Research output: Contribution to journalArticle

2518 Scopus citations

Abstract

Gene splicing by overlap extension is a new approach for recombining DNA molecules at precise junctions irrespective of nucleotide sequences at the recombination site and without the use of restriction endonucleases or ligase. Fragments from the genes that are to be recombined are generated in separate polymerase chain reactions (PCRs). The primers are designed so that the ends of the products contain complementary sequences. When these PCR products are mixed, denatured, and reannealed, the strands having the matching sequences at their 3' ends overlap and act as primers for each other. Extension of this overlap by DNA polymerase produces a molecule in which the original sequences are 'spliced' together. This technique is used to construct a gene encoding a mosaic fusion protein comprised of parts of two different mouse class-I major histocompatibility genes. This simple and widely applicable approach has significant advantages over standard recombinant DNA techniques.

Original languageEnglish (US)
Pages (from-to)61-68
Number of pages8
JournalGene
Volume77
Issue number1
DOIs
StatePublished - Apr 15 1989

Keywords

  • Genetic engineering
  • Taq polymerase
  • complementary sequences
  • exon
  • frequency of errors
  • intron
  • mosaic fusion protein
  • mouse histocompatibility genes
  • polymerase chain reaction
  • recombinant DNA

ASJC Scopus subject areas

  • Genetics

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