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Two-step hybridization and capture of a polynucleotide

  • US 6,534,273 B2
  • Filed: 09/18/2001
  • Issued: 03/18/2003
  • Est. Priority Date: 05/02/1997
  • Status: Expired due to Term
First Claim
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1. A method for amplifying a target polynucleotide that is present in a sample, comprising the steps of:

  • a. providing a capture probe that hybridizes specifically to a target polynucleotide present in a sample;

    b. providing a first primer oligonucleotide that hybridizes specifically to the target polynucleotide at a location different than the location that the capture probe hybridizes to the target polynucleotide;

    c. mixing the capture probe and the primer oligonucleotide with a sample suspected of containing the target polynucleotide at a first hybridization condition that favors hybridization of the capture probe to the target polynucleotide and the primer oligonucleotide to the target polynucleotide, thereby producing a capture probe;

    target polynucleotide;

    primer oligonucleotide complex formed by hybridization of the capture probe and the primer oligonucleotide to the target polynucleotide, wherein the first hybridization condition disfavors hybridization of an immobilized probe and the capture probe;

    d. providing an immobilized probe that hybridizes specifically to the capture probe at a second hybridization condition that is different from the first hybridization condition;

    e. incubating a mixture containing the capture probe;

    target polynucleotide;

    primer oligonucleotide complex and the immobilized probe at a second hybridization condition that favors hybridization of the immobilized probe and the capture probe, thereby producing a captured target polynucleotide complex consisting essentially of an immobilized probe;

    capture probe;

    target polynucleotide;

    primer oligonucleotide complex formed by hybridization of the immobilized probe to the capture probe in the capture probe;

    target polynucleotide;

    primer oligonucleotide complex;

    f. purifying the captured target polynucleotide complex from other sample components by washing the captured target polynucleotide complex, thereby producing a purified target polynucleotide retained in the immobilized probe;

    capture probe;

    target polynucleotide;

    primer oligonucleotide complex; and

    g. then amplifying a base sequence in the target polynucleotide by initiating nucleic acid polymerization from the first primer oligonucleotide using the target polynucleotide as a template and a DNA polymerase activity, thereby producing an amplified nucleic acid.

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