US4882269A
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Amplified hybridization assay
Assignee: Princeton University
•
Filing: 1986-12-11
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Granted: 1989-11-21
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Expires: 2006-12-11
Abstract
Abstract An amplified hybridization assay is described in which a family of signal-generating secondary probes bind to a primary probe that hybridizes to the target sequence of interest. Thus, an enormously amplified signal is generated by the hybridization event. The assay can be used for a variety of laboratory and clinical purposes and is automatable.
Inventors
- 👤 Robert J. Schneider
- 👤 Thomas E. Shenk
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-26 08:03
CPC Classifications
C; C07; C07H; C07H21/00; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6813; C12Q1/6816; C12Q1/682; Y; Y10; Y10S; Y10S435/00; Y10S435/803; Y10S435/81; Y10S436/00; Y10S436/80; Y10S436/805; Y10S436/807; Y10S436/808
Claims Analysis
Total Claims: 65
Claim 1
Independent Claim
141 words
A method for the detection of a target nucleotide sequence, comprising: (a) contacting the target nucleotide under conditions that permit hybridization with (i) a primary probe which comprises a polynucleotide sequence that is complementary to the target nucleotide sequence and a polymeric tail that has binding sites that are incapable of binding to the target sequence, and (ii) a plurality of secondary probes comprising a family of signal-generating probes each member of which comprises a signal generating component and a polymer capable of binding to a different portion of the tail of the primary probe; and (b) detecting the amplified signal generated by a reaction product formed in step (a), in which the polynucleotide sequence of the primary probe is hybridized to the target nucleotide and a plurality of secondary probes are bound to different portions of the primary probe tail.
Claim 2
Independent Claim
141 words
A method for the detection of a target nucleotide sequence, comprising: (a) contacting the target nucleotide under conditions that permit hybridization with (i) a primary probe which comprises a polynucleotide sequence that is complementary to the target nucleotide sequence and a polymeric tail that has binding sites that are incapable of binding to the target sequence, and (ii) a plurality of secondary probes comprising a family of signal-generating probes each member of which comprises a signal generating component and a polymer capable of binding to a different portion of the tail of the primary probe; and (b) detecting the amplified signal generated by a reaction product formed in step (a), in which the polynucleotide sequence of the primary probe is hybridized to the target nucleotide and a plurality of secondary probes are bound to different portions of the primary probe tail.
Claim 3
Dependent Claim
18 words
The method according to claim 1 in which the tail of the primary probe comprises a synthetic polymer.
Claim 4
Dependent Claim
18 words
The method according to claim 1 in which the tail of the primary probe comprises a natural polymer.
Claim 5
Dependent Claim
19 words
The method according to claim 1 in which the tail of the primary probe comprises a single-stranded nucleotide polymer.
Claim 6
Dependent Claim
14 words
The method according to claim 4 in which the nucleotide polymer comprises deoxyribonucleic acid.
Claim 7
Dependent Claim
14 words
The method according to claim 4 in which the nucleotide polymer comprises ribonucleic acid.
Claim 8
Dependent Claim
15 words
The method according to claim 1 in which the secondary probe comprises a synthetic polymer.
Claim 9
Dependent Claim
15 words
The method according to claim 1 in which the secondary probe comprises a natural polymer.
Claim 10
Dependent Claim
15 words
The method according to claim 1 in which the secondary probe comprises a nucleotide polymer.
Claim 11
Dependent Claim
14 words
The method according to claim 9 in which the secondary probe comprises deoxyribonucleic acid.
Claim 12
Dependent Claim
14 words
The method according to claim 9 in which the secondary probe comprises ribonucleic acid.
Claim 13
Dependent Claim
19 words
The method according to claim 1 in which the signal generating component of the secondary probe comprises a chromophore.
Claim 14
Dependent Claim
20 words
The method according to claim 1 in which the signal generating component of the secondary probe comprises a radioactive compound.
Claim 15
Dependent Claim
25 words
The method according to claim 1 in which the signal generating component of the secondary probe comprises an enzyme-substrate system that generates a detectable product.
Claim 16
Dependent Claim
12 words
The method according to claim 14 in which the enzyme comprises beta-galactosidase.
Claim 17
Dependent Claim
13 words
The method according to claim 14 in which the enzyme comprises alkaline phosphatase.
Claim 18
Dependent Claim
20 words
The method according to claim 1 in which the signal generating component of the secondary probe comprises a fluorescent compound.
Claim 19
Dependent Claim
20 words
The method according to claim 17 in which the fluorescent compound comprises an etheno-derivative of a single-stranded nucleic acid molecule.
Claim 20
Dependent Claim
25 words
The method according to claim 1 in which the signal generating component comprises a reporter group indirectly attached to the secondary probe via an agent.
Claim 21
Dependent Claim
21 words
The method according to claim 19 in which the reporter group is attached to avidin and the secondary probe is biotinylated.
Claim 22
Dependent Claim
25 words
The method according to claim 19 in which the secondary probe comprises a single-stranded nucleic acid molecule and the agent comprises a single-strand binding protein.
Claim 23
Dependent Claim
17 words
The method according to claim 1 in which the tail of the primary probe comprises a heteropolynucleotide.
Claim 24
Dependent Claim
15 words
The method according to claim 1 in which the secondary probe comprises a signal-generating heteropolynucleotide.
Claim 25
Dependent Claim
20 words
The method according to claim 12, 13, 14, 17 or 20 in which the secondary probe comprises a signal-generating heteropolynucleotide.
Claim 26
Dependent Claim
124 words
A hybridization assay kit for the detection of a target nucleotide sequence, comprising: (a) a primary probe which comprises a polynucleotide sequence that is complementary to the target nucleotide sequence and a polymeric tail that has binding sites that are incapable of binding to the target sequence; and (b) a plurality of secondary probes comprising a family of signal-generating probes each member of which comprises a signal-generating component and a polymer capable of binding to a different portion of the tail of the primary probe, which provides for the generation of an amplified signal when the polynucleotide sequence of the primary probe is hybridized to the target nucleotide and the plurality of secondary probes are bound to different portions of the primary probe tail.
Claim 27
Dependent Claim
124 words
A hybridization assay kit for the detection of a target nucleotide sequence, comprising: (a) a primary probe which comprises a polynucleotide sequence that is complementary to the target nucleotide sequence and a polymeric tail that has binding sites that are incapable of binding to the target sequence; and (b) a plurality of secondary probes comprising a family of signal-generating probes each member of which comprises a signal-generating component and a polymer capable of binding to a different portion of the tail of the primary probe, which provides for the generation of an amplified signal when the polynucleotide sequence of the primary probe is hybridized to the target nucleotide and the plurality of secondary probes are bound to different portions of the primary probe tail.
Claim 28
Dependent Claim
19 words
The kit according to claim 25 in which the tail of the primary probe comprises a single-stranded nucleotide polymer.
Claim 29
Dependent Claim
14 words
The kit according to claim 26 in which the nucleotide polymer comprises ribonucleic acid.
Claim 30
Dependent Claim
14 words
The kit according to claim 26 in which the nucleotide polymer comprises deoxyribonucleic acid.
Claim 31
Dependent Claim
15 words
The kit according to claim 25 in which the secondary probe comprises a natural polymer.
Claim 32
Dependent Claim
15 words
The kit according to claim 25 in which the secondary probe comprises a nucleotide polymer.
Claim 33
Dependent Claim
14 words
The kit according to claim 30 in which the secondary probe comprises deoxyribonucleic acid.
Claim 34
Dependent Claim
14 words
The kit according to claim 30 in which the secondary probe comprises ribonucleic acid.
Claim 35
Dependent Claim
19 words
The kit according to claim 25 in which the signal generating component of the secondary probe comprises a chromophore.
Claim 36
Dependent Claim
20 words
The kit according to claim 25 in which the signal generating component of the secondary probe comprises a radioactive compound.
Claim 37
Dependent Claim
25 words
The kit according to claim 25 in which the signal generating component of the secondary probe comprises an enzyme-substrate system that generates a detectable product.
Claim 38
Dependent Claim
12 words
The kit according to claim 35 in which the enzyme comprises beta-galactosidase.
Claim 39
Dependent Claim
13 words
The kit according to claim 35 in which the enzyme comprises alkaline phosphatase.
Claim 40
Dependent Claim
20 words
The kit according to claim 25 in which the signal generating compound of the secondary probe comprises a fluorescent compound.
Claim 41
Dependent Claim
20 words
The kit according to claim 38 in which the fluorescent compound comprises an etheno-derivative of a single-stranded nucleic acid molecule.
Claim 42
Dependent Claim
25 words
The kit according to claim 25 in which the signal generating component comprises a reporter group indirectly attached to the secondary probe via an agent.
Claim 43
Dependent Claim
21 words
The kit according to claim 40 in which the reporter group is attached to avidin and the secondary probe is biotinylated.
Claim 44
Dependent Claim
24 words
The kit according to claim 40 in which the secondary probe comprises a single-stranded nucleic acid molecule and the agent comprises single-strand binding protein.
Claim 45
Dependent Claim
18 words
The kit according to claim 25 in which the tail of the primary probe comprises a natural polymer.
Claim 46
Dependent Claim
18 words
The kit according to claim 25 in which the tail of the primary probe comprises a synthetic polymer.
Claim 47
Dependent Claim
15 words
The kit according to claim 25 in which the secondary probe comprises a synthetic polymer.
Claim 48
Dependent Claim
19 words
The hybridization assay kit according to claim 25 in which the tail of the primary probe comprises a heteropolynucleotide.
Claim 49
Dependent Claim
17 words
The hybridization assay kit according to claim 25 in which the secondary probe comprises a signal-generating heteropolynucleotide.
Claim 50
Dependent Claim
22 words
The hybridization assay kit according to claim 33, 34, 35, 38 or 41 in which the secondary probe comprises a signal-generating heteropolynucleotide.
Claim 51
Dependent Claim
131 words
A hybridization assay kit for the detection of a target nucleotide sequence, comprising: (a) a primary probe cassette which comprises a cloning vector having (i) a multiple cloning site into which a target nucleotide sequence can be inserted and cloned and (ii) nucleotide sequences which are capable of hybridizing to their complements which comprise a plurality of secondary probes; and (b) the plurality of secondary probes comprising a family of signal-generating probes each member of which comprises a signal-generating component and a nucleotide sequence capable of hybridizing to a different portion of the portion of the primary probe described in (a)(ii), which provides for the generation of an amplified signal when the plurality of secondary probes are hybridized to different portions of the portion of the primary probe described in (a)(ii).
Claim 52
Dependent Claim
131 words
A hybridization assay kit for the detection of a target nucleotide sequence, comprising: (a) a primary probe cassette which comprises a cloning vector having (i) a multiple cloning site into which a target nucleotide sequence can be inserted and cloned and (ii) nucleotide sequences which are capable of hybridizing to their complements which comprise a plurality of secondary probes; and (b) the plurality of secondary probes comprising a family of signal-generating probes each member of which comprises a signal-generating component and a nucleotide sequence capable of hybridizing to a different portion of the portion of the primary probe described in (a)(ii), which provides for the generation of an amplified signal when the plurality of secondary probes are hybridized to different portions of the portion of the primary probe described in (a)(ii).
Claim 53
Dependent Claim
19 words
The hybridization assay kit according to claim 49 in which the tail of the primary probe comprises a heteropolynucleotide.
Claim 54
Dependent Claim
25 words
The kit according to claim 49 in which the signal generating component comprises a reporter group indirectly attached to the secondary probe via an agent.
Claim 55
Dependent Claim
21 words
The kit according to claim 51 in which the reporter group is attached to avidin and the secondary probe is biotinylated.
Claim 56
Dependent Claim
24 words
The kit according to claim 51 in which the secondary probe comprises a single-stranded nucleic acid molecule and the agent comprises single-strand binding protein.
Claim 57
Dependent Claim
17 words
The hybridization assay kit according to claim 49 in which the secondary probe comprises a signal-generating heteropolynucleotide.
Claim 58
Dependent Claim
19 words
The kit according to claim 49 in which the signal generating component of the secondary probe comprises a chromophore.
Claim 59
Dependent Claim
20 words
A kit according to claim 49 in which the signal generating component of the secondary probe comprises a radioactive compound.
Claim 60
Dependent Claim
20 words
The kit according to claim 49 in which the signal generating compound of the secondary probe comprises a fluorescent compound.
Claim 61
Dependent Claim
20 words
The kit according to claim 57 in which the fluorescent compound comprises an etheno-derivative of a single-stranded nucleic acid molecule.
Claim 62
Dependent Claim
25 words
The kit according to claim 49 in which the signal generating component of the secondary probe comprises an enzyme-substrate system that generates a detectable product.
Claim 63
Dependent Claim
12 words
The kit according to claim 59 in which the enzyme comprises beta-galactosidase.
Claim 64
Dependent Claim
13 words
The method according to claim 59 in which the enzyme comprises alkaline phosphatase.
Claim 65
Dependent Claim
22 words
The hybridization assay kit according to claim 55, 56, 59, 57 or 52 in which the secondary probe comprises a signal-generating heteropolynucleotide.
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USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1992-11-20 | 1993-05-22 | 1993-11-21 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1996-11-20 | 1997-05-22 | 1997-11-21 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2000-11-20 | 2001-05-22 | 2001-11-21 | $7700.00 | Window Closed / Lapsed |