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Multiplex nucleic acid reactions

  • US 9,850,536 B2
  • Filed: 11/13/2014
  • Issued: 12/26/2017
  • Est. Priority Date: 02/07/2000
  • Status: Expired due to Term
First Claim
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1. A method for detecting target DNA sequences of different target DNA molecules in a sample, each of the target DNA sequences comprising, from 3′

  • to 5′

    ;

    contiguous first target domain, second target domain, and third target domain, comprising;

    (a) providing a sample having the different target DNA molecules;

    (b) contacting the sample with at least 100 different probe sets, thereby forming hybridization complexes with the different target DNA molecules, wherein each of the probe sets comprises;

    (i) a first probe comprising, from 5′

    to 3′

    ;

    a first universal priming sequence and a sequence that is substantially complementary to the first target domain;

    (ii) an intervening probe comprising a sequence that is complementary to the second target domain; and

    (iii) a second probe comprising 5′

    to 3′

    ;

    a sequence substantially complementary to the third target domain, and a second universal priming sequence,wherein the first universal priming sequence in each of the different probe sets is the same and wherein the second universal priming sequence in each of the different probe sets is the same;

    (c) capturing the hybridization complexes on a first solid support;

    (d) washing the first solid support to remove probes of the different probe sets that are not hybridized in the hybridization complexes;

    (e) forming extended first probes and extended intervening probes by contacting the hybridization complexes on first solid support with an extension enzyme and nucleotides;

    (f) forming amplification templates by ligating the extended first probes to the extended intervening probes, and ligating the extended intervening probes to the second probes;

    (g) amplifying the amplification templates with first and second universal primers that are complementary to the first universal priming sequence and the second universal priming sequence, thereby producing amplicons; and

    (h) capturing the amplicons on a second solid support that is different from the first solid support and detecting the amplicons on the surface of the second solid support, thereby detecting the target DNA sequences.

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