Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA probes and libraries.

DOI Web Site 83 Citations
  • L Diatchenko
    CLONTECH Laboratories, Inc., Palo Alto, CA 94303, USA.
  • Y F Lau
    CLONTECH Laboratories, Inc., Palo Alto, CA 94303, USA.
  • A P Campbell
    CLONTECH Laboratories, Inc., Palo Alto, CA 94303, USA.
  • A Chenchik
    CLONTECH Laboratories, Inc., Palo Alto, CA 94303, USA.
  • F Moqadam
    CLONTECH Laboratories, Inc., Palo Alto, CA 94303, USA.
  • B Huang
    CLONTECH Laboratories, Inc., Palo Alto, CA 94303, USA.
  • S Lukyanov
    CLONTECH Laboratories, Inc., Palo Alto, CA 94303, USA.
  • K Lukyanov
    CLONTECH Laboratories, Inc., Palo Alto, CA 94303, USA.
  • N Gurskaya
    CLONTECH Laboratories, Inc., Palo Alto, CA 94303, USA.
  • E D Sverdlov
    CLONTECH Laboratories, Inc., Palo Alto, CA 94303, USA.
  • P D Siebert
    CLONTECH Laboratories, Inc., Palo Alto, CA 94303, USA.

Description

<jats:p>A new and highly effective method, termed suppression subtractive hybridization (SSH), has been developed for the generation of subtracted cDNA libraries. It is based primarily on a recently described technique called suppression PCR and combines normalization and subtraction in a single procedure. The normalization step equalizes the abundance of cDNAs within the target population and the subtraction step excludes the common sequences between the target and driver populations. In a model system, the SSH technique enriched for rare sequences over 1,000-fold in one round of subtractive hybridization. We demonstrate its usefulness by generating a testis-specific cDNA library and by using the subtracted cDNA mixture as a hybridization probe to identify homologous sequences in a human Y chromosome cosmid library. The human DNA inserts in the isolated cosmids were further confirmed to be expressed in a testis-specific manner. These results suggest that the SSH technique is applicable to many molecular genetic and positional cloning studies for the identification of disease, developmental, tissue-specific, or other differentially expressed genes.</jats:p>

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