A modified overlap extension PCR method to create chimeric genes in the absence of restriction enzymes

Abstract

A modified overlap extension technique for the creation of chimeric genes is described: the method consists in three PCR steps. The first step is a conventional PCR reaction, in which oligonucleotide primers are partially complementary at their 5' ends to the adjacent fragments that are fused to create the chimer. The second PCR step consists in the fusion of the PCR fragments generated in the first step using the complementary extremities of the primers. The third step corresponds to the PCR amplification of the fusion product. The final PCR product is a chimeric gene built up with the different amplified PCR fragments. The technique is illustrated by the construction of a chimeric 5- hydroxytryptamine (5-HT, serotonin)1B/D receptor by combining one part of the human 5-HT1B (h5-HT1B) and two parts of the h5-HT1D receptor gene. The chimeric gene expressed in Cos-7 cells yielded similar binding properties as the wild type h5-HT1D receptor. © Rapid Science Ltd. 1998

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References

  1. Gelfand, D.H. and White, T.J. (1990). Thermostable DNA polymerases. In: PCR protocols: A Guide to Methods and Applications. MA Innis, DH Galfand, JJ Sninsky and TJ White, eds pp 129-141, Academic Press, New York.

    Google Scholar 

  2. Ho, S.N., Hunt, H.D., Horton, R.M., Pullen, J.K. and Pease, L.R. (1989). Gene 77:51-59.

    Google Scholar 

  3. Horton, R.M. (1995). Mol. Biotechnol. 3:93-99.

    Google Scholar 

  4. Horton, R.M., Hunt, H.D., Ho, S.N., Pullen, J.K. and Pease, L.R. (1989). Gene. 77:61-68.

    Google Scholar 

  5. Lo, K.M., Jones, S.S., Hackett, N.R. and Peace, L.R. (1984). Proc. Natl. Acad. Sci. USA 81:2285-2289.

    Google Scholar 

  6. Majumder, K. (1992). Gene. 110:89-94.

    Google Scholar 

  7. Pauwels, P.J., Reihsaus, E., Palmier, C., Journot, L., Lyons, J. and Colpaert, F.C. (1995). Cell. Pharmacol. 2:183-191.

    Google Scholar 

  8. Sanger, F., Nicklen, S. and Coulson, A.R. (1977). Proc. Natl. Acad. Sci. USA. 74:5463-5467.

    Google Scholar 

  9. Villarreal, X.C. and Long, G.L. (1991). Anal. Biochem. 197:362-367.

    Google Scholar 

  10. Weinshank, R.L., Zgombick, J.M., Macchi, M.J., Branchek, T.A. and Hartig, P.L. (1992). Proc. Natl. Acad. Sci. USA. 89:3630-3634.

    Google Scholar 

  11. Wychowsky, C., Emerson, S.U., Silver, J. and Feinstone, S.M. (1990). Nucleic Acids Res. 8:913-918.

    Google Scholar 

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Wurch, T., Lestienne, F. & Pauwels, P.J. A modified overlap extension PCR method to create chimeric genes in the absence of restriction enzymes. Biotechnology Techniques 12, 653–657 (1998). https://doi.org/10.1023/A:1008848517221

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Keywords

  • Serotonin
  • Oligonucleotide Primer
  • Binding Property
  • Fusion Product
  • Chimeric Gene