PCR Protocols pp 287-304 | Cite as

Generation of a Polymerase Chain Reaction Renewable Source of Subtractive cDNA

  • W. Michael Kuehl
  • James Battey
Part of the Methods in Molecular Biology book series (MIMB, volume 15)


Differential (+/-) first-strand cDNA screening methods identify clones corresponding to mRNAs that are expressed at a higher level in one of a pair of phenotypically different cells. This approach is limited by the fact that screening of libraries with labeled first-strand cDNAs synthesized from unfractionated mRNA can detect clones containing sequences representing approx 0.1% or more of the complexity of mRNA (i.e., mRNAs present at greater than about 200 copies per cell since a typical mammalian cell line contains approx 250,000 mRNAs).


Adapter Sequence Microfuge Tube Subtractive cDNA Subtractive cDNA Library Parental mRNA 
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  1. 1.
    Alt, F. W., Kellems, R. E., Bertino, J. R., and Schimke, R. T. (1978) Selective multiplication of dihydrofolate reductase genes in methotrexate-resistant variant of cultured murine cells. J. Biol. Chem. 163, 1357–1370.Google Scholar
  2. 2.
    Timberlake, W. E. (1980) Developmental gene regulation in Aspergillus nidulans. Dev. Biol. 78, 497–510.PubMedCrossRefGoogle Scholar
  3. 3.
    Sargent, T. D. and Dawid, I. B. (1983) Differential gene expression in the gastrula of Xenopus laevis. Science 222, 135–139.PubMedCrossRefGoogle Scholar
  4. 4.
    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
  5. 5.
    Klickstein, L. B. (1987) Production of a subtractive cDNA library, in Current Protocols in Molecular Biology, vol. 1 (Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. E., Seidman, J. G., Smith, J. A., and Struhl, K., eds.), Wiley, New York, pp. 5.8.6.–5.8.13.Google Scholar
  6. 6.
    Duguid, J. R., Rohwer, R. G., and Seed, B. (1988) Isolation of cDNAs of scrapiemodulated RNAs by subtractive hybridization of a cDNA library. Proc. Natl. Acad. Sci. USA 85, 5738–5742.PubMedCrossRefGoogle Scholar
  7. 7.
    Travis, G. H. and Sutcliffe, J. G. (1988) Phenol emulsion-enhanced DNAdriven subtractive cDNA cloning: isolation of low-abundance monkey cortexspecific mRNAs. Proc. Natl. Acad. Sci. USA 85, 1696–1700.PubMedCrossRefGoogle Scholar
  8. 8.
    Palazzolo, M. J. and Meyerowitz, E. M. (1987) A family of lambda phage cDNA cloning vectors, lambda SWAJ, allowing the amplification of RNA sequences. Gene 52, 197–206.PubMedCrossRefGoogle Scholar
  9. 9.
    Rubenstein, J. L. R., Brice, A. E. J., Ciaranello, R. D., Denney, D., Porteus, M. H., and Usdin, T. B. (1990) Subtractive hybridization system using single-stranded phagemids with directional inserts. Nucleic Acids Res. 18, 4833–4842.PubMedCrossRefGoogle Scholar
  10. 10.
    Batra, S. K., Metzgar, R. S., and Hollingsworth, M. A. (1991) A simple, effective method for the construction of subtracted cDNA libraries. GATA 8, 129–133.Google Scholar
  11. 11.
    Timblin, C., Battey, J., and Kuehl, W. M. (1990) Application for PCR technology to subtractive cDNA cloning: identification of genes expressed specititally in murine playmacytoma cells. Nucleic Acids Res. 18, 1587–1593.PubMedCrossRefGoogle Scholar
  12. 12.
    Wieland, I., Bolger, G., Asouline, G., and Wigler, M. (1990) A method for difference cloning: Gene amplification following subtractive hybridization. Proc. Natl. Acad. Sci USA 87, 2720–2724.PubMedCrossRefGoogle Scholar
  13. 13.
    Britten, R. J., Graham, D. E., and Neufeld, B. R. (1974) Analysis of repeating DNA sequences by reassociation. Methods Enzymol. 29, 363–418.PubMedCrossRefGoogle Scholar
  14. 14.
    Galau, G. A., Briten, R. J., and Davidson, E. H. (1977) Studies on nucleic acid reassociation kinetics: rate of hybridization of excess RNA with DNA, compared to the rate of DNA renaturation. Proc. Natl. Acad. Sci. USA 74, 1020–1023.PubMedCrossRefGoogle Scholar
  15. 15.
    Van Ness, J. and Hahn, W. E. (1982) Physical parameters affecting the rate and completion of RNA driven hybridization of DNA: new measurements relevant to quantitation based on kinetics. Nucleic. Acids Res. 10, 8061–8077.PubMedCrossRefGoogle Scholar
  16. 16.
    Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Separation of single-stranded and double-stranded DNA by hydroxyapatite chromatography, in Molecular Cloning: A Laboratory Manual, vol. 3. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp. E.30–E.33.Google Scholar
  17. 17.
    Frohman, M. A., Dush, M. K., and Martin, G. R. (1988) Rapid production of full-length cDNAs from rare transcripts: Amplification using a single genespecific oligonucleotide primer. Proc. Natl. Acad. Sci USA 85, 8998–9002.PubMedCrossRefGoogle Scholar
  18. 18.
    Wang, Z. and Brown, D. D. (1991) A gene expression screen. Proc. Natl. Acad. Sci. USA 88, 11,505–11,509.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 1993

Authors and Affiliations

  • W. Michael Kuehl
    • 1
  • James Battey
    • 1
  1. 1.NCI-Navy Medical OncologyBethesda

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