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Detectably labeled dual conformation oligonucleotide probes, assays and kits

  • US 5,925,517 A
  • Filed: 05/12/1995
  • Issued: 07/20/1999
  • Est. Priority Date: 11/12/1993
  • Status: Expired due to Term
First Claim
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1. A signaling unitary hybridization probe useful in an assay having conditions that include a detection temperature for detecting at least one nucleic acid strand containing a preselected nucleic acid target sequence, said probe comprising:

  • a single-stranded target complement sequence having from 10 to about 140 nucleotides, having a 5'"'"' terminus and a 3'"'"' terminus, and being complementary to the target sequence;

    flanking the target complement sequence, a pair of oligonucleotide arms consisting of a 5'"'"' arm sequence covalently linked to said 5'"'"' terminus and a 3'"'"' arm sequence covalently linked to said 3'"'"' terminus, said pair of oligonucleotide arms forming a stem duplex 3-25 nucleotides in length, said stem duplex having a melting temperature above said detection temperature under said assay conditions; and

    at least one interacting label pair, each pair comprising a first label moiety conjugated to the 5'"'"' arm sequence and a second label moiety conjugated to the 3'"'"'arm sequence,said probe having, under said assay conditions in the absence of said target sequence, a characteristic signal whose level is a function of the degree of interaction of said first and second label moieties, said signal having a first level at 10°

    C. below said melting temperature, a second level at 10°

    C. above said melting temperature and a third level at said detection temperature,wherein under the assay conditions at the detection temperature and in the presence of an excess of said target sequence, hybridization of the target complement sequence to the target sequence alters the level of said characteristic signal from said third level toward the second level by an amount of at least ten percent of the difference between the first and second levels.

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