US4725536A
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Reagent polynucleotide complex with multiple target binding regions, and kit and methods
Assignee: Genetics Institute LLC
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Filing: 1985-09-19
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Granted: 1988-02-16
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Expires: 2005-09-19
Abstract
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.
Inventors
- 👤 Edward F. Fritsch
- 👤 Mary Collins
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-25 10:54
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6876; C12Q1/6883; C12Q1/6813; C12Q1/6816; C12Q1/6823; C12Q1/6827; Y; Y10; Y10S; Y10S435/00; Y10S435/81
Claims Analysis
Total Claims: 24
Claim 1
Independent Claim
126 words
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.
Claim 2
Dependent Claim
126 words
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.
Claim 3
Dependent Claim
27 words
The reagent complex of claim 1 wherein the selected nucleotide sequence is present in a biological sample in varying degrees of proximity to the target nucleotide sequence.
Claim 4
Dependent Claim
26 words
The reagent complex of claim 2 wherein the target nucleotide sequence and selected nucleotide sequence are chromosomal DNA sequences whose relative position changes with disease states.
Claim 5
Dependent Claim
30 words
The reagent complex of claim 3 wherein the target nucleotide sequence and selected nucleotide sequence are normally on separate chromosomes but, in a disease state, are on a common chromosome.
Claim 6
Dependent Claim
30 words
The reagent complex of claim 3 wherein the target nucleotide sequence and selected nucleotide sequence are normally on a common chromosome but, in a disease state, are on separate chromosomes.
Claim 7
Dependent Claim
27 words
The reagent complex of claim 3 wherein the target nucleotide sequence is a constant region and the selected nucleotide sequence is a variable region of chromosomal DNA.
Claim 8
Dependent Claim
19 words
The reagent complex of claim 2 wherein the target nucleotide sequence and selected nucleotide sequence are messenger RNA sequences.
Claim 9
Dependent Claim
19 words
A diagnostic kit comprising the reagent complex of claim 1 and a polynucleotide reagent containing the selected nucleotide sequence.
Claim 10
Dependent Claim
16 words
The diagnostic kit of claim 8 wherein the probe polynucleotide is immobilized in the reagent complex.
Claim 11
Dependent Claim
30 words
The diagnostic kit of claim 8 wherein the probe polynucleotide contains a moiety immobilized by an affinity reagent and the kit further comprises an immobilized affinity reagent for the moiety.
Claim 12
Dependent Claim
40 words
The diagnostic kit of claim 8 wherein the target nucleotide sequence and selected nucleotide sequence are present on a common polynucleotide strand in samples from normal individuals, but are present on separate polynucleotide strands in the event of a translocation.
Claim 13
Dependent Claim
39 words
The reagent complex of claim 1 wherein the probe polynucleotide has a third target nucleotide sequence and the labeled polynucleotide is further bound by complementary base pair binding to at least a portion of the third target nucleotide sequence.
Claim 14
Independent Claim
209 words
A method for the determination of a predetermined target nucleotide sequence in the nucleic acid of a biological sample comprising the steps: (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.
Claim 15
Independent Claim
209 words
A method for the determination of a predetermined target nucleotide sequence in the nucleic acid of a biological sample comprising the steps: (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.
Claim 16
Dependent Claim
15 words
The method of claim 13 wherein the probe polynucleotide is immobilized in the reagent complex.
Claim 17
Dependent Claim
29 words
The method of claim 13 wherein the reagent complex is in solution during the contacting step (I) and the probe polynucleotide contains a moiety immobilizable by an affinity reagent.
Claim 18
Dependent Claim
27 words
The method of claim 15 wherein the washing step (II) comprises first immobilizing the reagent complex with an immobilized affinity reagent for the moiety and then washing.
Claim 19
Independent Claim
248 words
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: (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.
Claim 20
Independent Claim
248 words
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: (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.
Claim 21
Dependent Claim
33 words
The method of claim 17 wherein the molar ratio of reagent complex to either the anticipated level of target nucleotide sequence or selected nucleotide sequence, whichever is higher, is at least about 100:1.
Claim 22
Dependent Claim
19 words
The method of claim 17 wherein the ratio is between about 10 3 :1 and about 10 6 :1.
Claim 23
Dependent Claim
25 words
The method of claim 16 wherein the target nucleotide sequence and selected nucleotide sequence are chromosomal DNA sequences whose relative position changes with disease states.
Claim 24
Dependent Claim
18 words
The method of claim 16 wherein the target nucleotide sequence and selected nucleotide sequence are messenger RNA sequences.
References Cited (Backward Citations)
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| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1991-02-15 | 1991-08-17 | 1992-02-16 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1995-02-15 | 1995-08-17 | 1996-02-16 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1999-02-15 | 1999-08-17 | 2000-02-16 | $7700.00 | Window Closed / Lapsed |