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The use of the polymerase chain reaction in plant transformation studies

Summary

Transformed root lines of Nicotiana species, containing NPTII and Gus genes, were used to study the parameters affecting the use of the Polymerase Chain Reaction as a routine analytical tool for quickly analysing plant transformants for the presence of a foreign gene. The basic reaction mix as described by Cetus Corporation (Saiki 1989) was close to optimal for successful PCR amplification of internal sequences of both NPTII and Gus from genomic plant DNA. The temperature of primer annealing in the PCR protocols was found to be the most important variable, as low temperatures caused amplification of artefact bands and smearing after analysis on ethidium bromide agarose gels. Various formulae for calculating the Tm for binding of primers of various lengths (20–30 bases) are described in relation to predicting suitable annealing temperatures in the PCR.

For tobacco species the PCR reaction worked efficiently with up to 2 μg of genomic DNA. However, with DNA from Mentha species (mint), an inhibitor of the PCR process was co-extracted with the DNA which prevented amplification of target sequences, if more than 10 ng of genomic DNA was present in the reaction.

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Abbreviations

c7dCTP:

7-deaza-2′-deoxyguanosine

GUS-β-Glucuronidase, NPT II:

neomycin phosphotransferase II

PCR:

polymerase chain reaction

TBE:

Tris Borate

EtBr:

ethidium bromide

References

  1. Beck, E., Ludwig, G., Averswald, E.A., Reiss, B., Schaller, H. (1982) Gene 19: 327–336.

  2. Bennett, M.D. (1972) Proc. R. Soc. London B. 181, 109–135.

  3. Bevan, M.W. (1984) Nuc. Acid Res. 12: 8711–8721.

  4. Dellaporta, S.L. (1983) Plant Mol. Biol. Rep. 1: 19–21.

  5. Depicker, A., Stachel, S., Dhaese, P., Zambryski, P., Goodman, H.M. (1982) J. Mol. Appl. Genet. 1: 561–574.

  6. Erlich, H.A. (Ed.) (1989) PCR Technology: Principles and applications for DNA amplifications. Stockton Press, New York, London, Tokyo, Melbourne, Hong Kong.

  7. Fonzi, W.A. and Sypherd, P.S. (1987) J. Biol. Chem. 262: 10127–10135.

  8. Furner, I.J., Huffman, G.A., Amasino, R.M., Garfinkel, D.J., Gordon, M.P., Nester, E.W. (1986). Nature 319: 422–427.

  9. Goldberg, S.B., Flick, J.S., Rogers, S.G. (1984). Nucleotide sequence of the tmr locus of Agrobacterium tumefaciens pTiT37 T-DNA. Nucleic Acids Res. 12: 4665–77.

  10. Haff, L.A. and Mezei, L.M. (1989) Perkin Elmer Cetus Amplifications 1, 8–10.

  11. Hamill, J.D., Prescott, A., Martin, C. (1987) Plant Mol. Biol. 9: 573–584.

  12. Hamill, J.D., Robins, R.J., Rhodes, M.J.C. (1989) Planta Medica 55, 354–357.

  13. Hamill, J.D., Robins, R.J., Parr, A.J., Evans, D.M., Furze, J.M., Rhodes, M.J.C. (1990) Plant Mol. Biol. 15, 27–38.

  14. Hirayama, T., Muranaka, T., Ohkawa, H., Oka, A. (1988) Mol. Gen. Genet. 213, 229–237

  15. Innis, M.A. and Gelfand, D.H. (1990) in PCR protocols: A guide to methods and applications. Academic Press Inc., San Diego, New York, London, Sydney, Tokyo, Toronto. Innis, M.A., Gelfand, D.H., Sninsky, J.J. and White, T.J. (eds.), pp. 3–12.

  16. Jefferson, R.A., Kavanagh, T.A., Bevan, M.W. (1987) EMBO J 6: 3901–3907.

  17. Lassner, M.W., Peterson, P., Yoder, J.I. (1989) Plant Mol. Biol. Reporter 7, 116–127.

  18. Maniatis, T., Fritsch, E.F., Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual. Cold Spring Harbour, New York.

  19. Powell, B. S., Powell, G. K., Morris, R. O., Rogowsky, P. M., Kado, C. I. (1987) Mol. Microbiol. 1: 309–316.

  20. Saiki, R.K. and Gelfand, D.H. (1989). Perkin Elmer Cetus Amplifications 1, 4–5.

  21. Saiki, R.K. (1989) In PCR Technology. Erlich, H.A. (ed.) Stockton Press, p7–16.

  22. Saiki, R.K. (1990) In PCR Protocols: Innis, M.A., Gelfand, D.H., Sinisky, J.T., White, T.J. (eds.) Academic Press Inc., p13–20.

  23. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning, 2nd Edition, Cold Spring Harbour Laboratory Press

  24. Spencer, A., Hamill, J.D., Rhodes, M.J.C. (1990) Plant Cell Rep. 8: 601–604.

  25. Thein, S.L. and Wallace, R.B. (1986). In Human gentic disorders: a practical approach (ed. K.E. Davis), p33–50 IRL Press, Herndon, Virginia.

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Communicated by W. Barz

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Hamill, J.D., Rounsley, S., Spencer, A. et al. The use of the polymerase chain reaction in plant transformation studies. Plant Cell Reports 10, 221–224 (1991). https://doi.org/10.1007/BF00232562

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Keywords

  • Ethidium Bromide
  • Foreign Gene
  • Basic Reaction
  • Internal Sequence
  • Transformation Study