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Methods for identifying low-abundance polynucleotides and related compositions

  • US 20040006033A1
  • Filed: 06/30/2003
  • Published: 01/08/2004
  • Est. Priority Date: 08/06/2001
  • Status: Abandoned Application
First Claim
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1. A subtractive hybridization method for identifying one or more polynucleotides in a test sample that are absent from, or less abundant in, a reference sample, said method comprising:

  • a) providing high-abundance enriched polynucleotide strands of, or prepared from, a pool of test or reference polynucleotides that is enriched in high-abundance polynucleotide sequences relative to a test or reference polynucleotide sample, respectively;

    b) contacting the high-abundance enriched polynucleotide strands with test polynucleotide strands of, or prepared from, the test polynucleotide sample under hybridization conditions to form a first hybridization mixture, thereby producing unhybridized test polynucleotide strands that are enriched in low-abundance polynucleotide sequences relative to the test polynucleotide sample;

    c) synthesizing polynucleotide strands from the unhybridized test polynucleotide strands, thereby producing low-abundance enriched test polynucleotide strands;

    d) providing low-abundance-enriched reference polynucleotide stands of, or prepared from, a reference pool of polynucleotides that is enriched in low-abundance polynucleotide sequences relative to the reference polynucleotide sample;

    e) contacting the low-abundance enriched reference polynucleotide strands with the low-abundance enriched test polynucleotide strands under hybridization conditions to form a second hybridization mixture, thereby producing hybrid duplexes, unhybridized low-abundance enriched reference polynucleotide strands, and unhybridized low-abundance enriched test polynucleotide strands;

    f) removing or digesting the hybrid duplexes; and

    g) producing test-specific duplexes from the unhybridized low-abundance enriched test polynucleotide strands.

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