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Processes for detecting or quantifying more than one nucleic acid in a library

  • US 8,597,888 B2
  • Filed: 07/29/2004
  • Issued: 12/03/2013
  • Est. Priority Date: 06/30/2001
  • Status: Expired due to Term
First Claim
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1. A process for detecting or quantifying an individual nucleic add sequence of interest in a library comprising the steps of:

  • a) providing;

    (i) an array of fixed or immobilized nucleic acids identical or complementary in part or whole to sequences of said nucleic acids of interest;

    (ii) a library of nucleic acid analytes which may contain the individual nucleic acid sequence of interest sought to be detected or quantified;

    (iii) polymerizing means for synthesizing nucleic acid copies of said nucleic acid analytes, said polymerizing means comprising a first set of primers, a second set of primers, and a polymerase, wherein said first set of primers are fixed or immobilized to a solid support, and wherein said second set of primers comprises at least one production center;

    (iv) a set of double-stranded oligonucleotides or polynucleotides comprising a sequence complementary to at least one segment or sequence of said second set of primers; and

    (v) means for ligating said set of oligonucleotides or polynucleotides (iv);

    b) contacting said library of nucleic acid analytes provided in step a) ii) with said first set of primers to form more than one first bound entity;

    c) extending by means of said polymerase the first set of primers in said more than one first bound entity formed in step b) by means of template sequences provided by said nucleic acid analytes to form first copies of said analytes;

    d) blunt end ligating said set of double-stranded oligonucleotides or polynucleotides a) (iv) to the 3′

    end of said first copies of said analytes formed in step c) to form a double-stranded ligated product;

    d′

    ) removing said nucleic acid analytes from said double-stranded ligated product, thereby rendering said first copies of said analytes into single-stranded form;

    e) contacting said ligated product formed in step d′

    ) with said second set of primers provided in step a) iii) to form more than one second bound entity, wherein said second bound entity is formed by hybridization of said second set of primers to said ligated set of oligonuclebtides or polynucleotides in said ligated product;

    f) extending by means of said polymerase the second set of primers in said more than one second bound entity formed in step e) by means, of template sequences provided by said ligated products formed in step d) to form more than one complex comprising said ligated products and said extended second set of primers;

    g) synthesizing under isothermal or isostatic conditions one or more nucleic acid copies from a production center in said second set of primers in said complexes formed in step f);

    h) hybridizing said nucleic acid copies formed in step g) to said array of nucleic acids provided in step a) (i); and

    i) detecting or quantifying any of said hybridized copies obtained in step h).

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