Solution phase nucleic acid sandwich assay and polynucleotide probes useful therein

Claim 2

A method according to claim 1, wherein said nucleic acid sequence complementary to said capturing reagent recognition sequence is bound directly to said separation means, and said analyte, (a), (b), and (c) are combined together for a time sufficient for nucleic acid complexes to form, followed by the addition of said nucleic acid sequence complementary to said capturing reagent recognition sequence bound to said separation means.

Claim 3

A method according to claim 1, wherein said separation means is a solid support and wherein said capturing reagent is bound thereto.

Claim 4

An assay method for detecting a nucleic acid sequence in a sample, employing two sets of reagents: a first labeling set; and a second capturing set, said method comprising: combining in a liquid medium under binding conditions for complementary nucleic acid sequences, said sample containing analyte in single-stranded form, members of said labeling set of reagents comprising: (a) a plurality of labeling nucleic acid probes including a fluorescer or enzyme label, different probes having a different first analyte complementary sequence and a first noncomplementary region comprising a label reagent recognition sequence, said first analyte complementary sequence having a nucleic acid sequence about 15 to 100 nucleotides in length and said first noncomplementary region optionally being at most about 5kb in length; and (b) a labeling reagent having a nucleic acid sequence complementary to said label reagent recognition sequence; and members of said second capturing set of reagents comprising: (c) a plurality of capturing nucleic acid probes, different probes having a different second analyte complementary sequence and a second noncomplementary region comprising a capturing reagent recognition sequence, said second analyte complementary sequence having a nucleic acid sequence about 15 to about 100 nucleotides in length and said noncomplementary region optionally being at most about 5kb in length; and (d) a capturing reagent having a nucleic acid sequence complementary to said capturing reagent recognition sequence; binding said capturing reagent to a solid support through a specific binding pair comprising a hapten and an antibody, either during or after said combining; separating said label into a bound phase and an unbound phase by means of said solid support; and detecting the amount of bound or unbound label as determinative of the presence of said analyte.

Claim 5

A kit for detecting a nucleic acid analyte comprising: (1) members of a labeling set of reagents comprising: (a) a plurality of labeling nucleic acid probes, different probes having a different first analyte complementary sequence and a first noncomplementary region comprising a label reagent recognition sequence, said first analyte complementary sequence having a nucleic acid sequence about 15 to 100 nucleotides in length and said first noncomplementary region optionally being at most about 5 kb in length; and (b) a labeling reagent having a nucleic acid sequence complementary to said label reagent recognition sequence, wherein said label provides, directly or indirectly, a detectable signal; and (2) members of a second capturing set of reagents comprising: (c) a plurality of capturing nucleic acid probes, different probes having a different second analyte complementary sequence and a second noncomplementary region comprising a second capturing reagent recognition sequence having a nucleic acid sequence about 15 to about 100 nucleotides in length and said second noncomplementary region optionally being at most about 5 kb in length; (d) a capturing reagent having a nucleic acid sequence complementary to said capturing reagent recognition sequence a first member of a specific binding pair conjugate; and (e) support means conjugated to a second complementary member of said specific binding pair.

Claim 6

The use of the following in the manufacture of a kit for detecting a nucleic acid sequence: (1) members of a labeling set of reagents comprising: (a) a plurality of labeling nucleic acid probes, different probes having a different first analyte complementary sequence and a first noncomplementary region comprising a label reagent recognition sequence, said first analyte complementary sequence having a nucleic acid sequence about 15 to 100 nucleotides in length and said first noncomplementary region optionally being at most about 5 kb in length; and (b) a labeling reagent having a nucleic acid sequence complementary to said label reagent recognition sequence, wherein said label provides, directly or indirectly, a detectable signal; and (2) members of a second capturing set of reagents comprising: (c) a plurality of capturing nucleic acid probes, different probes having a different second analyte complementary sequence and a second noncomplementary region comprising a second capturing reagent recognition sequence having a nucleic acid sequence about 15 to about 100 nucleotides in length and said second noncomplementary region optionally being at most about 5kb in length; (d) a capturing reagent having a nucleic acid sequence complementary to said second recognition sequence and containing a first member of a specific binding pair conjugate; and (e) support means conjugated to a second complementary member of said specific binding pair.

Claim 7

The use of a polynucleotide probe in an assay method according to any one of claims 1 to 4, wherein the probe has at least two nucleotides, at least one of which is given by the structure wherein R¹ is a reactive group derivatized with a detectable label, R² is an optional linking moiety including an amide, thioether or disulfide linkage or a combination thereof, R³ is selected from the group consisting of hydrogen, methyl, bromine, fluorine and iodine, R⁶ is H, OH or OR where R is a protecting group and x is an integer in the range of 1 to 8 inclusive.

Claim 8

The use according to claim 6 wherein a polynucleotide probe as defined in claim 7 is employed.