Molecular Biotechnology

, Volume 3, Issue 2, pp 129–134

Selection of primers for polymerase chain reaction

  • Wojciech Rychlik
Protocol

Abstract

One of the most important factors affecting the quality of PCR is the choice of primers. In general, the longer the PCR product the more difficult it is to select efficient primers and set appropriate designing primers, and in general, the more DNA sequence information is available, the better the ch0ance of finding an optimal primer pair. Efficient primers can be designed by avoiding the following flaws: primer-dimer formation, self-complementarity, too lowTm of the primers, and/or their incorrect internal stability profile. Tips on subcloning PCR products, calculating duplex stability (predicting dimer formation strength), and designing degenerate primers are given.

Index entries

PCR primer design primer internal stability 

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References

  1. 1.
    Breslauer, K. J., Frank, R., Blocker, H., and Markey, L. A. (1986) Predicting DNA duplex stability from the base sequence.Proc. Natl. Acad. Sci. USA 83, 3746–3750.PubMedCrossRefGoogle Scholar
  2. 2.
    Freier, S. M., Kierzek, R., Jaeger, J. A., Sugimoto, N., Caruthers, M. H., Neilson, T., and Turner, D. H. (1986) Improved free-energy parameters for predictions of RNA duplex stability.Proc. Natl. Acad. Sci. USA 83, 9373–9377.PubMedCrossRefGoogle Scholar
  3. 3.
    Groebe, D. R. and Uhlenbeck, O. C. (1988) Characterization of RNA hairpin loop stability.Nucleic Acids Res. 16, 11,725–11,735.CrossRefGoogle Scholar
  4. 4.
    Cheng, S., Fockler, C., Barnes, W. M., and Russell, H. (1994) Effective amplification of long targets from clones inserts and human genomic DNA.Proc. Natl. Acad. Sci. USA 91, 5696–5699.Google Scholar
  5. 5.
    Wu, D. Y., Ugozzoli, L., Pal, B. K., Qian, J., and Wallace, R. B. (1991) The effect of temperature and oligonucleotide primer length on the specificity and efficiency of amplification by the polymerase chain reaction.DNA Cell Biol.,10, 233–238.PubMedCrossRefGoogle Scholar
  6. 6.
    Rychlik, W., Spencer, W. J., and Rhoads, R. E. (1990) Optimization of the annealing temperature for DNA amplification in vitro.Nucleic Acids Res. 18, 6409–6412.PubMedCrossRefGoogle Scholar
  7. 7.
    Rychlik, W. and Rhoads, R. E. (1989) A computer program for choosing optimal oligonucleotides for filter hybridization, sequencing and in vitro amplification of DNA.Nucleic Acids Res. 17, 8543–8551.PubMedCrossRefGoogle Scholar
  8. 8.
    Lee, C. C. and Caskey, C. T. (1990) cDNA cloning using degenerate primers, inPCR Protocols ( Innis, M. A., Gelfand, D. H., Sninsky, J. J., and White, T. J. eds.), Academic, New York, pp. 46–53.Google Scholar
  9. 9.
    Kwok, S., Kellogg, D. E., McKinney, N., Spasic, D., Goda, L., Levenson, C., and Sninsky, J. J. (1990) Effects of primer-template mismatches on the polymerase chain reaction: human immunodeficiency virus type 1 model studies.Nucleic Acids Res. 18, 999–1005.PubMedCrossRefGoogle Scholar
  10. 10.
    Eckert, K. A. and Kunkel, T. A. (1990) High fidelity DNA synthesis by theThermus aquaticus DNA polymerase.Nucleic Acids Res. 18, 3739–3744.PubMedCrossRefGoogle Scholar
  11. 11.
    Petruska, J., Goodman, M. F., Boosalis, M. S., Sowers, L. C., Cheong, C., and Tinoco, I., Jr. (1988) Comparison between DNA melting thermodynamics and DNA polymerase fidelity.Proc. Natl. Acad. Sci. USA 85, 6252–6256.PubMedCrossRefGoogle Scholar
  12. 12.
    Kawasaki, E. (1990) Amplification of RNA, inPCR Protocols (Innis, M. A., Gelfand, D. H., Sninsky, J.J., and White, T. J. eds.), Academic, New York, pp. 21–27.Google Scholar
  13. 13.
    White, B. A., ed. (1993)Methods in Molecular Biology, vol. 15: PCR Protocols, Humana, Totowa, NJ.Google Scholar
  14. 14.
    New England BioLabs, 1993–1994 Catalog, “Cleavage close to the end of DNA fragments,” p. 180.Google Scholar
  15. 15.
    Jung, V., Pestka, S. B., and Pestka, S. (1990) Efficient cloning of PCR generated DNA containing terminal restriction endonuclease recognition sites.Nucleic Acids Res. 18, 6156.PubMedCrossRefGoogle Scholar
  16. 16.
    Eckert, K. A. and Kunkel, T. A. (1991) The fidelity of DNA polymerase used in PCR, inPolymerase Chain Reaction: A Practical Approach ( McPherson, M. J., Quirke, P., and Taylor, G.R. eds.), IRL, Oxford, UK, pp. 227–246.Google Scholar
  17. 17.
    Marchuk, D., Drumm, M., Saulino, A., and Collins, F. S. (1991) Construction of T-vectors, a rapid and general system for direct cloning of unmodified PCR productsNucleic Acids Res. 19, 1154.PubMedCrossRefGoogle Scholar
  18. 18.
    Holton, T. A. and Graham, M. W. (1991) A simple and efficient method for direct cloning of PCR products using ddT-tailed vectorsNucleic Acids Res. 19, 1156.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 1995

Authors and Affiliations

  • Wojciech Rychlik
    • 1
  1. 1.National Biosciences Inc.Plymouth

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