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Capture primers and capture sequence linked solid supports for molecular diagnostic tests

  • US 9,416,409 B2
  • Filed: 07/30/2010
  • Issued: 08/16/2016
  • Est. Priority Date: 07/31/2009
  • Status: Expired due to Fees
First Claim
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1. A method comprising;

  • a) contacting a sample suspected of containing a target nucleic acid with a capture primer and a reverse primer, wherein said capture primer comprises;

    i) a 3′

    region configured to hybridize to said target nucleic acid such that it can be extended by a polymerase, and ii) a 5′

    region comprising a capture sequence; and

    wherein said contacting is under conditions such that;

    i) said 3′

    region of said capture primer hybridizes to said target nucleic acid and is extended to generate a first amplification product, andii) said reverse primer hybridizes to said first amplification product and is extended to generate a second amplification product, wherein said second amplification product comprises a 3′

    capture sequence complement capable of hybridizing to said capture sequence; and

    b) treating said sample under conditions such that said second amplification product is separated from said first amplification product;

    c) contacting said second amplification product with a solid support comprising a plurality of bound capture sequences under conditions such that said 3′

    capture sequence complement of said second amplification product hybridizes to one of said bound capture sequences to generate a hybridized solid support; and

    d) treating said hybridized solid support under conditions such that one of said bound capture sequences is extended along said second amplification product to generate a target sequence that is linked to said solid support; and

    e) treating said target sequence linked to said solid support under conditions such that at least part of the nucleic acid sequence of said target sequence is determined by a method comprising;

    i) contacting said target sequence with at least one nucleotide incorporating biocatalyst, labeled nucleotides, and at least one primer nucleic acid that is at least partially complementary to at least a subsequence of said target sequence, under conditions whereby said nucleotide incorporating biocatalyst extends said primer nucleic acid to produce an extended primer nucleic acid by incorporating said labeled nucleotides at a terminal end of said extended primer nucleic acid, wherein nucleotides that comprise different nucleobases comprise different labels, wherein the different labels produce detectable signals as or after the labeled nucleotides are incorporated at the terminal end of the extended primer nucleic acid, which detectable signals identify the labeled nucleotides incorporated at the terminal end of the extended primer nucleic acid and/or complementary nucleotides in the template nucleic acid, and wherein the detectable signals are detected as or after the labeled nucleotides are incorporated at the terminal end of the extended primer nucleic acid; and

    ii) determining said nucleic acid sequence of said target sequence by a method selected from;

    pyrosequencing, sequencing-by-synthesis, sequencing-by-ligation, nanopore sequencing, single molecule SBS, and real-time sequencing.

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