to BioTechniques free email alert service to receive content updates.
Gene Splicing by Overlap Extension: Tailor-Made Genes Using the Polymerase Chain Reaction
Robert M. Horton, Zeling Cai, Steffan N. Ho, and Larry R. Pease
Full Text (PDF)

Synthetic Uses of PCR

Several modifications to the basic SOE concept are possible. A “mega-primer” approach which uses fewer primers has recently been used by several groups (14, 16). Recently, Sandhu, Precup and Kline (personal communication) have demonstrated that fragments can be SOEn directly to a vector, and then recircularized by blunt-end ligation. Thus it may be possible to entirely avoid the use of restriction enzymes for the construction of recombinant genes in appropriate vectors.Different approaches to using PCR as a synthetic tool are also being developed. For example, asymmetric PCR has been used to generate what is in effect a huge mutagenic oligonucleotide for use in an otherwise standard m13- based mutagenesis strategy, in which regions as large as an exon can be replaced (4).

Limitations of SOE

The major drawback of the gene SOEing technique is that, even though the frequency of polymerase errors is low, it is necessary to sequence the cloned products in order to be certain that you have what you want. This limits the usefulness of gene SOEing to engineering problems which are not conveniently solved by more conventional methods. One caveat to bear in mind is that, in addition to mediating directed recombination, PCR can lead to random recombinations between related genes present in the same reaction (12). A partially elongated fragment of one gene can act as a primer on a different gene, producing a recombinant product. This can be viewed as either a hazard in attempting to amplify one gene out of a multi-gene family or a possible means of generating a set of recombinants between related molecules.


PCR is much more than a sensitive tool for detecting DNA sequences. Splicing by Overlap Extension, or “gene SOEing,” is a novel, PCR-mediated recombinant DNA technology. It represents a significant advancement over the standard restriction enzymebased methods for recombining genes because it does not rely on restriction sites. Thus it allows much finer control of recombination for genetic engineering. Also, the sequence of the overlap region is determined by the primer design, making it possible to perform mutagenesis and recombination simultaneously. This technically simple and rapid approach is entirely different from methods now used for generating modified and recombinant DNA fragments, and we believe it marks the beginning of a new generation of recombinant DNA technology.


R.M.H and Z.C. were supported by predoctoral fellowships from the Mayo Graduate School, Mayo Foundation, and S.N.H. was supported by a Medical Scientist Scholarship from the Life and Health Insurance Research Fund. We would like to thank Drs. Henry Hunt and Jeffrey Pullen for sharing their gene SOEing experiences with us.

1.) Benoist, C.O., D.J. Mathis, M.R. Kanter, V.E. Williams, and IIand H.O. McDevitt. 1983. Regions of allelic hypervariability in the murine Aa. immune response gene. Cell 34:169-177.

2.) Dulau, L., A. Cheyrou, and M. Aigle. 1989. Directed mutagenesis using PCR. Nucleic Acids Res. 17:2873.

3.) Estess, P., A.B. Begovich, M. Koo, P.P. Jones, and H.O. McDevitt. 1986. Sequence analysis and structure-function correlations of murine q, k, u, s, and f haplotype I-A eDNA clones. Proc. Natl. Acad. Sci. USA 83:3594-3598.

4.) Feinstone, S., C. Wychowski, S. Emerson, and J. Silver. 1989. Synthesis of chimeric hepatitis A virus/poliovirus subgenomic eDNA by a PCR mutagenesis system. J. Cell. Biol. Supplement (abstract #WH130) 13E:283.

5.) Higuchi, R., B. Krummel, and R.K. Saiki. 1988. A general method of in vitro preparation and specific mutagenesis of DNA fragments: study of protein and DNA interactios. Nucleic Acids Res. 15:7351-7367.

6.) Ho, S.N., H.D. Hunt, R.M. Horton, J.K. Pullen, and L.R. Pease. 1989. Site-directed mutagenesis by overlap extension using the Polymerase Chain Reaction. Gene 77:51-59.

7.) Horton, R.M., H.D. Hunt, S.N. Ho, J.K. Pullen, and L.R. Pease. 1989. Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene 77:61-68.

8.) Kadowaki, H., T. Kadowaki, F.E. Wondisfordand, and S.I. Taylor. 1989. Use of Polymerase Chain Reaction catalyzed by Taq DNA polymerase for site-specific mutagenesis. Gene 76:161-166.

9.) Kraft, T., J. Tardiff, K.S. Krauter, and L.A. Leinwand. 1988. Using mini-prep plasmid DNA for sequencing double stranded templates with Sequenase™. BioTechniques 6:544-547.

10.) Mullis, K., and F. Faloona. 1987. Specific synthesis of DNA in vitro via a polymerasecatalyzed Chain Reaction. Methods Enzymol. 155:335-350.

11.) llis, K., F. Faloona, S. Scharf, R. Saiki, G. Horn, and H. Erlich. 1986. Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction. Cold Spr. Harb. Symp. Quant. Bioi. L1:263-273.

12.) Paabo, S., R.G. Higuchi, and A.C. Wilson. 1989. Ancient DNA and the polymerase chain reaction: the emerging field of molecular archaeology. J. Biol. Chem. 264:9709-9712.

13.) Pullen, J.K., H.D. Hunt, R.M. Horton, and L.R. Pease. 1989. The functional significance of two amino acid polymorphisms in the antigen-presenting domain of class I MHC molecules: molecular dissection of Kbm3. J. Immunol. 143:1674-1679.

14.) Sarkar, G., and S.S. Sommer. 1990. The “megaprimer” method of site-directed mutagenesis. BioTechniques 8:404-407.

15.) Suggs, S.V., T. Hirose, T. Miyake, E.H. Kawashima, M.J. Johnson, K. Itakura, and R.B. Wallace. 1981.Use of synthetic oligodeoxyribonucleotides for the isolation of specific cloned DNA sequences. In D.D. Brown, and C.F. Fox (Eds.) Developmental Biology Using Purified Genes. Academic Press, New York:683-693.

16.) Yon, J., and M. Fried. 1989. Precise gene fusion by PCR. Nucleic Acids Res. 17:4895.

  1    2    3