Reagent polynucleotide complex with multiple target binding regions, and kit and methods
First Claim
1. A reagent complex for the determination of a predetermined target nucleotide sequence in the nucleic acid of a biological sample comprising:
- (a) a probe polynucleotide having;
(i) a first target binding region which is capable of complementary base pair binding via hydrogen bonds of purine/pyrimidine base pairs to a target nucleotide sequence; and
(ii) a second target binding region which is capable of complementary base pair binding via hydrogen bonds of purine/pyrmimdine base pairs to a selected nucleotide sequence; and
(b) a labeled polynucleotide which is bound by complementary base pair binding via hydrogen bonds of purine/pyrimidine base pairs to the probe polynucleotide in at least a portion of the first target binding region and in at least a portion of the second target binding region.
1 Assignment
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Accused Products
Abstract
Reagent complexes containing a probe polynucleotide with at least two target binding regions. A labeled polynucleotide is bound to at least a portion of each target binding region. In one method, sample nucleic acid displaces labeled polynucleotide from one target binding region; after washing, a reagent polynucleotide displaces labeled polynucleotide from the other target binding region. In a second method, sample nucleic acid strands having two target nucleic acid sequences in proximity (e.g., sequences translocated in certain cancers) displace the labeled polynucleotide from both target binding region.
224 Citations
21 Claims
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1. A reagent complex for the determination of a predetermined target nucleotide sequence in the nucleic acid of a biological sample comprising:
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(a) a probe polynucleotide having; (i) a first target binding region which is capable of complementary base pair binding via hydrogen bonds of purine/pyrimidine base pairs to a target nucleotide sequence; and (ii) a second target binding region which is capable of complementary base pair binding via hydrogen bonds of purine/pyrmimdine base pairs to a selected nucleotide sequence; and (b) a labeled polynucleotide which is bound by complementary base pair binding via hydrogen bonds of purine/pyrimidine base pairs to the probe polynucleotide in at least a portion of the first target binding region and in at least a portion of the second target binding region. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12)
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13. A method for the determination of a predetermined target nucleotide sequence in the nucleic acid of a biological sample comprising the steps:
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(I) contacting the sample with a reagent complex comprising; (a) a probe polynucleotide having; (i) a first target binding region which is capable of complementary base pair binding via hydrogen bonds of purine/pyrimidine base pairs to the target nucleotide sequence; and (ii) a second target binding region which is capable of complementary base pair binding via hydrogen bonds of purine/pyrimidine base pairs to a selected nucleotide sequence; and (b) a labeled polynucleotide which is bound by complementary base pair binding via hydrogen bonds of purine/pyrimidine base pairs to the probe polynucleotide in at least a portion of the first target binding region and in at least a portion of the second target nucleotide sequence, under conditions in which the target nucleotide sequence, if present, displaces the labeled polynucleotide from the first target binding region; (II) washing the reagent complex; (III) further contacting the washed reagent complex with a polynucleotide reagent comprising the selected nucleotide sequence under conditions in which the selected nucleotide sequence displaces the labeled polynucleotide from the second target binding region; and (IV) detecting labeled polynucleotide displaced from the first and second target binding regions. - View Dependent Claims (14, 15, 16, 20, 21)
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17. A method for determining the presence of a target nucleotide sequence and a selected nucleotide sequence within a common polynucleotide strand of the nucleic acid of a biological sample, which comprises the steps:
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(I) contacting the sample with a reagent complex comprising; (a) a probe polynucleotide having; (i) a first target binding region which is capable of complementary base pairing binding via hydrogen bonds of purine/pyrimidine base pairs to the target nucleotide sequence; and (ii) a second target binding region which is capable of complementary base pair binding via hydrogen bonds of purine/pyrimidine base pairs to a selected nucleotide sequence; and (b) a labeled polynucleotlde which is bound by complementary base pair binding via hydrogen bonds of purine/pyrimidine base pairs to the probe polynucleotide in at least a portion of the first target binding region and in at least a portion of the second target nucleotide sequence; under conditions, including sufficient molar excess of reagent complex relative to anticipated levels of the target nucleotide sequence and of the selected nucleotide sequence, under which sample nucleic acid strands having the target nucleotide sequence and the selected nucleotide sequence, if present within a common sample polynucleotide strand, will displace substantially more labeled polynucleotide strands from the first and second target binding regions of a common probe strand than will target nucleotide sequence and selected nucleotide sequence on separate sample polynucleotide strands; and (II) detecting labeled polynucleotide displaced from the first and second target binding regions of a common probe strand. - View Dependent Claims (18, 19)
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Specification