Displacement polynucleotide method and reagent complex employing labeled probe polynucleotide

Claim 1

A method for determining the presence and amount of a predetermined target nucleotide sequence in the nucleic acid of a biological sample which comprises the steps: (a) providing a reagent complex formed by a single nucleic acid strand comprising (i) labeled probe polynucleotide which is capable of base pair binding via hydrogen bonds of purine/pyrimidine bases to the target nucleotide sequence, and (ii) a second polynucleotide which is covalently attached by phosphate-sugar backbone to the labeled polynucleotide and which is bound by base pair binding via hydrogen bonds of purine/pyrimidine base pairs to the labeled probe polynucleotide in a region of the labeled probe polynucleotide at least partially coextensive with the region in which the labeled probe polynucleotide is capable of binding to the target nucleotide sequence; (b) contacting the reagent complex with a sample under conditions in which the target nucleotide sequence, if present, binds to the labeled probe polynucleotide and displaces second polynucleotide from the labeled probed polynucleotide; (c) immobilizing said displaced second polynucleotide by binding to an immobilized third polynucleotide complementary to a portion of the displaced second polynucleotide and capable of binding by hybridization to a portion of said displaced second polynucleotide, thereby separating labeled probe polynucleotides from which second polynucleotide has been displaced from intact reagent complexes; and (d) measuring the resulting immobilized labeled probe polynucleotide/target sequence hybrids.

Claim 2

A method for determining the presence and amount of a predetermined target nucleotide sequence in the nucleic acid of a biological sample which comprises the steps: (a) providing a reagent complex formed by a single nucleic acid strand comprising (i) labeled probe polynucleotide which is capable of base pair binding via hydrogen bonds of purine/pyrimidine bases to the target nucleotide sequence, and (ii) a second polynucleotide which is covalently attached by phosphate-sugar backbone to the labeled polynucleotide and which is bound by base pair binding via hydrogen bonds of purine/pyrimidine base pairs to the labeled probe polynucleotide in a region of the labeled probe polynucleotide at least partially coextensive with the region in which the labeled probe polynucleotide is capable of binding to the target nucleotide sequence; (b) contacting the reagent complex with a sample under conditions in which the target nucleotide sequence, if present, binds to the labeled probe polynucleotide and displaces second polynucleotide from the labeled probed polynucleotide; (c) immobilizing said displaced second polynucleotide by binding to an immobilized third polynucleotide complementary to a portion of the displaced second polynucleotide and capable of binding by hybridization to a portion of said displaced second polynucleotide, thereby separating labeled probe polynucleotides from which second polynucleotide has been displaced from intact reagent complexes; and (d) measuring the resulting immobilized labeled probe polynucleotide/target sequence hybrids.

Claim 5

A method for determining the presence and amount of a predetermined target nucleotide sequence in the nucleic acid of a biological sample which comprises the steps: (a) providing a reagent complex formed by a single nucleic acid strand comprising (i) labeled probe polynucleotide which is capable of base pair binding via hydrogen bonds of purine/pyrimidine bases to the target neucleotide sequence, and (ii) a second polynucleotide which is stably joined to the labeled polynucleotide and which is bound by base pair binding via hydrogen bonds of purine/pyrimidine base pairs to the labeled probe polynucleotide in a region of the labeled probe polynucleotide at least partially coextensive with the region in which the labeled probe polynucleotide is capable of binding to the target nucleotide sequence; (b) contacting the reagent complex with a sample under conditions in which the target nucleotide sequence, if present, binds to the labeled probe polynucleotide and displaces second polynucleotide from the labeled probe polynucleotide; (c) immobilizing said displaced second polynucleotide by binding to an immobilized third polynucleotide complementary to a portion of the displaced second polynucleotide and capable of binding by hybridization to a portion of said displaced second polynucleotide, thereby separating labeled probe polynucleotides from which second polynucleotide has been displaced from intact reagent complexes; and (d) measuring the resulting immobilized labeled probe polynucleotide/target sequence hybrids.

Claim 6

A method for determining the presence and amount of a predetermined target nucleotide sequence in the nucleic acid of a biological sample which comprises the steps: (a) providing a reagent complex formed by a single nucleic acid strand comprising (i) labeled probe polynucleotide which is capable of base pair binding via hydrogen bonds of purine/pyrimidine bases to the target neucleotide sequence, and (ii) a second polynucleotide which is stably joined to the labeled polynucleotide and which is bound by base pair binding via hydrogen bonds of purine/pyrimidine base pairs to the labeled probe polynucleotide in a region of the labeled probe polynucleotide at least partially coextensive with the region in which the labeled probe polynucleotide is capable of binding to the target nucleotide sequence; (b) contacting the reagent complex with a sample under conditions in which the target nucleotide sequence, if present, binds to the labeled probe polynucleotide and displaces second polynucleotide from the labeled probe polynucleotide; (c) immobilizing said displaced second polynucleotide by binding to an immobilized third polynucleotide complementary to a portion of the displaced second polynucleotide and capable of binding by hybridization to a portion of said displaced second polynucleotide, thereby separating labeled probe polynucleotides from which second polynucleotide has been displaced from intact reagent complexes; and (d) measuring the resulting immobilized labeled probe polynucleotide/target sequence hybrids.