Detectably labeled dual conformation oligonucleotide probes, assays and kits

A unitary hybridization probe useful in an assay for the detection of at least one nucleic acid target containing a preselected target sequence, said probe being capable of assuming a closed conformation and an open conformation and comprising:

• a) a target complement sequence of from 10 to about 140 nucleotides complementary to said preselected nucleic acid target sequence, having a 5' terminus and a 3' terminus,
• b) an affinity pair comprising a first affinity moiety covalently linked to said 5' terminus and a second affinity moiety covalently linked to said 3' terminus, said affinity pair interacting sufficiently to hold said probe in the closed conformation in said assay in the absence of said nucleic acid target, and
• c) a label pair comprising a first label moiety conjugated to said first affinity moiety and a second label moiety conjugated to said second affinity moiety, said label moieties interacting to affect a measurable characteristic of at least one of them when said probe is in the closed conformation,
• wherein hybridization of said target complement sequence to said target sequence in said assay causes said probe to assume its open conformation, in which said label moieties do not so interact.

A labeled, single-stranded probe for detecting the presence or absence in a sample of a preselected nucleic acid target sequence in an assay having conditions including a detection temperature, said probe comprising:

• (a) an oligonucleotide sequence comprising
• (i) a target complement sequence perfectly complementary to the target sequence, said target complement sequence having 7 to 25 nucleotides, a 5' terminus, and a 3' terminus,
• (ii) a first arm sequence covalently linked to said 5' terminus and a second arm sequence covalently linked to said 3' terminus, said first arm sequence having 3 to 8 nucleotides immediately adjacent to said 5' terminus that are complementary to nucleotides of said second arm sequence immediately adjacent said 3' terminus, said arms forming a stem duplex having a melting temperature at least 5.degree. C. above said detection temperature under said assay conditions, said oligonucleotide sequence, when oppositely terminally labeled with a fluorescer/quencher pair producing under said conditions upon addition of excess model target, an increase in fluorescence that is at least 10 percent as great as the increase in fluorescence caused by heating it from 10.degree. C. below to 10.degree. C. above said melting temperature;
• and
• (b) at least one detectable label,
• wherein said probe produces a detectable signal above background in said assay of a sample containing an excess of said target sequence that is at least five times higher above background than in said assay of a sample containing an excess of a sequence differing from said target sequence by a single internally located nucleotide.

A signaling unimolecular 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 60 nucleotides, have 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 at least 5.degree. C. above said detection temperature under said assay conditions;
• and
• at least interacting label pair covalently linked to said target complement sequence,
• said probe having, under said assay conditions in the absence of said target sequence, a characteristic fluorescence signal whose level is a function of the degree of interaction of said label pair, said signal having a first level at 10.degree. C. below said melting temperature, a second level at 10.degree. 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 said second level by an amount of at least ten percent of the difference between the first and second levels.