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T-structure invasive cleavage assays, consistent nucleic acid dispensing, and low level target nucleic acid detection

  • US 8,354,232 B2
  • Filed: 07/20/2010
  • Issued: 01/15/2013
  • Est. Priority Date: 06/09/2006
  • Status: Active Grant
First Claim
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1. A method of target nucleic acid dependent amplification of a non-target sequence in a sample comprising;

  • a) incubating a sample with stem oligonucleotides, upstream oligonucleotides, primers, dNTPs, and a polymerase under conditions such that, if a target nucleic acid is present;

    i) a 3′

    target specific region of said stem oligonucleotides hybridizes to said target nucleic acid, and a stem region of said stem oligonucleotides remains available for hybridization to said upstream oligonucleotides,ii) a 5′

    target specific region of said upstream oligonucleotides hybridizes to said target nucleic acid, and a stem specific region of said upstream oligonucleotides hybridizes to a portion of said stem region of said stem oligonucleotides, andiii) said polymerase extends a 3′

    end of said upstream oligonucleotides using said stem region of said stem oligonucleotides as a template to generate extended upstream oligonucleotides that comprise an upstream oligonucleotide extended region;

    b) heating said sample to separate said extended upstream oligonucleotides from said stem oligonucleotides and said target nucleic acid;

    c) cooling said sample under conditions such that said primers hybridize to at least a portion of said upstream oligonucleotide extended region of said extended upstream oligonucleotides; and

    d) incubating said sample under conditions such that said primers are extended by said polymerase using said extended upstream oligonucleotides as templates such that stem amplicon sequences are generated.

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