Subtractive cDNA Cloning Using Magnetic Beads and PCR

  • Thierry G. Coche
Part of the Methods in Molecular Biology™ book series (MIMB, volume 67)

Abstract

Subtractive cloning is a method that facilitates the isolation of nucleotide sequences present in a test sample but absent or present at much lower levels, in a reference sample. A variety of situations lend themselves to this methodology and it has been used, for example, to characterize changes in levels of gene expression during development (1, 2, 3), in pathological situations (4, 5, 6), in inductive events (7,8), and for the isolation of tissue-specific genes (1,9, 10, 11). Current methodologies also allow detection of pathogens and integrated retroviruses present at exceedingly low levels in the cell (12).

Keywords

Magnesium Phenol EDTA Agarose Citrate 

References

  1. 1.
    Timblin, C., Battey, J., and Kuehl, M. W. (1990) Application of PCR technology to subtractive cDNA cloning. identification of genes expressed specifically in murine plasmacytome cells. Nucleic Acid Res. 18, 1587–1593.PubMedCrossRefGoogle Scholar
  2. 2.
    Sargent, T. D. and Dawid, I. B. (1983) Differential gene expression in the gastrula of Xenopus laevis. Science 222, 135–139.PubMedCrossRefGoogle Scholar
  3. 3.
    Mathiopoulos, C. and Sonenshein, A. L. (1989) Identification of Bacillus subtilis genes expressed early during sporulation. Mol. Microbiol. 3, 1071–1081.PubMedCrossRefGoogle Scholar
  4. 4.
    Bowes, C., Danciger, M., Kozak, C. A., and Farber, D. B. (1989) Isolation of a candidate cDNA for the gene causing retinal degeneration in the rd mouse. Proc. Natl. Acad. Sci. USA 86, 9722–9726.PubMedCrossRefGoogle Scholar
  5. 5.
    Reynet, C. and Kahn, R. C. (1993) Rad: a member of the Ras family overex-pressed in muscle of type II diabetic humans. Science 262, 1441–1444.PubMedCrossRefGoogle Scholar
  6. 6.
    Cole, S. P. C., Bhardwaj, G., Gerlach, J. H., Machie, J. E., Grant, C. E., Almquist, K. C., Stewart, A. J., Kurz, E U., Duncan, A. M. V., and Deeley, R. G. (1992) Over-expression of a transporter gene in a multidrug-resistant human lung cancer cell line. Science 258, 1650–1654.PubMedCrossRefGoogle Scholar
  7. 7.
    Nedivi, E., Hevroni, D., Naot, D., Israeli, D., and Citri, Y. (1993) Numerous candidate plasticity-related genes revealed by differential cloning. Nature 363, 718–721.PubMedCrossRefGoogle Scholar
  8. 8.
    Parfett, C. L., Hofbauer, R., Brudzynski, K., Edwards, D. R., and Denhardt, D. T. (1989) Differential screening of a cDNA library with probes amplified in a heterologous host: isolation of murine GRP78 (BiP) and other serum-regulated low abundance mRNAs. Gene 82, 291–303.PubMedCrossRefGoogle Scholar
  9. 9.
    Swaroop, A., Xu, J., Agarwal, N., and Weissman, S. M. (1991) A simple and efficient cDNA library substraction procedure: isolation of human retina-specific cDNA clones. Nucleic Acids Res. 19, 1954.PubMedCentralPubMedCrossRefGoogle Scholar
  10. 10.
    Hedrick, S. M., Cohen, D. I., Nielsen, E. A., and Davis, M. M. (1984) Isolation of cDNA clones encoding T cell-specific membrane-associated proteins. Nature 308, 149–153.PubMedCrossRefGoogle Scholar
  11. 11.
    Tedder, T. F., Streuli, M., Schlossman, S. F., and Saito, H. (1988) Isolation and structure of a cDNA encoding the B1 (CD20) cell-surface antigen of human B lymphocytes. Proc. Natl. Acad. Sci. USA 85, 208–212.PubMedCrossRefGoogle Scholar
  12. 12.
    Chang, Y., Cesarman, E., Pessin, M. S., Lee, F., Culpepper, J., Knowles, D. M., and Moore, P. S. (1994) Identification of herpesvirus-like DNA sequences in AIDS-associated Kaposi’s sarcoma. Science 266, 1865–1869.PubMedCrossRefGoogle Scholar
  13. 13.
    Stürzl, M. and Roth, W. K. (1990) PCR-synthesized single-stranded DNA: a use ful tool for ‘hyb’ and ‘HAP’ standardization for construction of subtraction libraries. Trends Genet. 6, 106.PubMedGoogle Scholar
  14. 14.
    Lebeau, M.-C., Alvarez-Bolado, G., Wahli, W., and Catsicas, S. (1991) PCR driven DNA-DNA competitive hybridization: a new method for sensitive differential cloning. Nucleic Acids Res. 19, 4778.PubMedCentralPubMedCrossRefGoogle Scholar
  15. 15.
    Klickstein, L. B. (1989) Production of a subtracted cDNA library, in Current Protocols in Molecular Biology (Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. D., Smith, J. A., and Struhl, K., eds.), John Wiley, New York, pp. 5.8.9–5.8.15.Google Scholar
  16. 16.
    Hampson, I. N., Pope, L., Cowling, G. J., and Dexter, T. M. (1992) Chemical crosslinking subtraction (CCLS): a new method for the generation of subtractive hybridization probes. Nuclezc Acids Res. 20, 2899.CrossRefGoogle Scholar
  17. 17.
    Lisitsyn, N., Lisitsyn, N., and Wigler, M. (1993) Cloning the difference between two complex genomes. Science 259, 946–951.PubMedCrossRefGoogle Scholar
  18. 18.
    Rodriguez, I. R and Chader, G. J. (1992) A novel method for the isolation of tissue-specific genes. Nucleic Acids Res. 20, 3528.PubMedCentralPubMedCrossRefGoogle Scholar
  19. 19.
    Schraml, P., Shipman, R., Stulz, P., and Ludwig, C. U. (1993) cDNA subtraction library construction using a magnet-assisted subtraction technique. Trends Genet. 9, 70, 71.PubMedCrossRefGoogle Scholar
  20. 20.
    Coche, T., Dewez, M., and Beckers, M.-C. (1994) Generation of an unlimited supply of a subtracted probe using magnetic beads and PCR. Nucleic Acids Res. 22, 1322, 1323.PubMedCentralPubMedCrossRefGoogle Scholar
  21. 21.
    Bröker, M. (1993) PCR-based constraction of subtractive cDNA library using magnetic beads. BioTechniques 4, 610, 611.Google Scholar
  22. 22.
    Britten, R. J. and Davidson, E. H. (1985) Hybridization strategy, in Nucleic Acid Hybridization. A Practical Approach (Hames, B. D. and Higgins, S. J., eds.), IRL, Oxford, UK, pp. 3–15.Google Scholar
  23. 23.
    Sambrook, J., Fritsch, E. F., and Maniatis, T., eds. (1989) Molecular Cloning A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.Google Scholar

Copyright information

© Humana Press Inc. 1997

Authors and Affiliations

  • Thierry G. Coche
    • 1
  1. 1.Organogenesis LabUniversity of OxfordUK

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