Subtractive Hybridization and Construction of cDNA Libraries

  • Bruce Blumberg
  • Juan Carlos Izpisúa Belmonte
Part of the METHODS IN MOLECULAR BIOLOGY™ book series (MIMB, volume 461)

1. Introduction

Genes that are differentially expressed both in time and space are the basis for how single cells, through the process of embryonic development, give rise to animals with an extraordinary diversity of cell types. As a first step in understanding differential gene expression, many researchers seek to identify those genes whose transcripts are temporally or spatially restricted to Particular cells, tissues, or embryonic stages. Although there are a variety of methods suitable for identifying moderately to highly expressed genes, the isolation of the most interesting class of mRNAs, those that are not abundant, but that may be cell-or tissue-specific, remains the most difficult task.

Several basic types of methods have been employed to identify low-abundance, tissue-specific transcripts. The more classical differential hybridization techniques (e.g., 1) are mostly limited to the detection of moderately abundant transcripts representing >0.05% of the mRNA population (2)....


Sodium Dodecyl Sulfate Isoamyl Alcohol Subtract cDNA Library Guanidine Thiocyanate Column Buffer 
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  1. 1.
    St. John, T. P. and Davis, R. W. (1979) Isolation of galactose-inducible DNA sequences from Saccharomyces cerevisiae by differential plaque filter hybridization. Cell 16, 443–452.CrossRefPubMedGoogle Scholar
  2. 2.
    Sargent, T. D. and Dawid, I. B. (1983) Differential gene expression in the gastrula of Xenopus laevis. Science 222, 135–139.CrossRefPubMedGoogle Scholar
  3. 3.
    Wang, Z. and Brown, D. D. (1991) A gene expression screen. Proc. Natl. Acad. Sci.USA 88, 11,505–11,509.Google Scholar
  4. 4.
    Aasheim, H. C., Deggerdal, A., Smeland, E. B., and Hornes, E. (1994) A simple subtraction method for the isolation of cell-specific genes using magnetic monodis-perse polymer Particles. Biotechniques 16, 716–721.PubMedGoogle Scholar
  5. 5.
    Hara, E., Kato, T., Nakada, S., Sekiya, S., and Oda, K. (1991) Subtractive cDNA cloning using oligo(dT)30-latex and PCR: isolation of cDNA clones specific to undif-ferentiated human embryonal carcinoma cells. Nucleic Acids Res. 19, 7097–7104.CrossRefPubMedGoogle Scholar
  6. 6.
    Li, W. B., Gruber, C. E., Lin, J. J., Lim, R., D'Alessio, J. M., and Jessee, J. A.(1994) The isolation of differentially expressed genes in fibroblast growth factor stimulated BC3H1 cells by subtractive hybridization. Biotechniques 16, 722–729.PubMedGoogle Scholar
  7. 7.
    Lopez-Fernandez, L. A. and del Mazo, J. (1993) Construction of subtractive cDNA libraries from limited amounts of mRNA and multiple cycles of subtraction. Bio-techniques 15, 654–659.Google Scholar
  8. 8.
    Sharma, P., Lonneborg, A., and Stougaard, P. (1993) PCR-based construction of subtractive cDNA library using magnetic beads. Biotechniques 15, 610–612.PubMedGoogle Scholar
  9. 9.
    Maniatis, T., Fritsch, E. F., and Sambrook, J. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N Y.Google Scholar
  10. 10.
    Han, J. H., Stratowa, C., and Rutter, W. J. (1987) Isolation of full-length putative rat lysophospholipase cDNA using improved methods for mRNA isolation and cDNA cloning. Biochemistry 26, 1617–1625.CrossRefPubMedGoogle Scholar
  11. 11.
    Blumberg, B., Wright, C. V. E., De Robertis, E. M., and Cho, K. W. Y. (1991) Organizer-specific homeobox genes in Xenopus laevis embryos. Science 253, 194–196.CrossRefPubMedGoogle Scholar
  12. 12.
    Forster, A. C., McInnes, J. L., Skingle, D. C., and Symons, R. H. (1985) Non-radioactive hybridization probes prepared by the chemical labelling of DNA and RNA with a novel reagent, photobiotin. Nucleic Acids Res. 13, 745–761.CrossRefPubMedGoogle Scholar
  13. 13.
    Sive, H. L. and St. John, T. (1988) A simple subtraction hybridization technique employing photoactivatable biotin and phenol extraction. Nucleic Acids Res. 16, 10,937.Google Scholar
  14. 14.
    Sasaki, Y. F., Ayusawa, D., and Oishi, M. (1994) Construction of a normalized cDNA library by introduction of a semi-solid mRNA-cDNA hybridization system.Nucleic Acids Res. 22, 987–992.CrossRefPubMedGoogle Scholar
  15. 15.
    Feinberg, A. P. and Vogelstein, B. (1983) A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132, 6–13.CrossRefPubMedGoogle Scholar
  16. 16.
    Church, G. M. and Gilbert, W. (1984) Genomic sequencing. Proc. Natl. Acad. Sci.USA 81, 1991–1995.CrossRefPubMedGoogle Scholar

Copyright information

© Humana Press, a Part of Springer Science + Business Media, LLC 2008

Authors and Affiliations

  • Bruce Blumberg
    • 1
  • Juan Carlos Izpisúa Belmonte
    • 2
  1. 1.Gene Expression Laboratory, Stem Cell Research Center, and Laboratory of GeneticsThe Salk Institute for Biological StudiesLa JollaUSA
  2. 2.The Center of Regenerative Medicine in BarcelonaBarcelonaSpain

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