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

doi:10.1016/0378-1119(89)90359-4

Gene

Volume 77, Issue 1, 15 April 1989, Pages 61-68

https://doi.org/10.1016/0378-1119(89)90359-4 Get rights and content

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.

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

Abstract

Gene

Methods Enzymol.

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Nature

Antiproliferative activity of a hybrid protein between interferongamma and tumor necrosis factor-beta

Science

A general method of in vitro preparation and specific mutagenesis of DNA fragments: study of protein and DNA interactions

Nucleic Acids Res.

Construction of a recombinant expression plasmid encoding a staphylococcal protein A-ricin A fusion protein

Gene