US6037130A
Expired / Lapsed
US
⏱️ 0.0 years remaining
Wavelength-shifting probes and primers and their use in assays and kits
Assignee: PAUL ROYALTY FUND LP
•
Filing: 1998-07-28
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Granted: 2000-03-14
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Expires: 2018-07-28
Abstract
Abstract Hairpin-forming oligonucleotide probes and primers triple-labeled with a pair of florophores, a shorter wavelength harvester and a longer wavelength emitter, and a quencher. When the probes and primers are stimulated by light that excites the harvester, opening causes an increase in fluorescence by the emitter, while fluorescence from the harvester is continually suppressed. The probes and primers may be used for detection of nucleic acid targets in assays, including amplification assays. Assay kits are provided.
Inventors
- 👤 Sanjay Tyagi
- 👤 Fred R. Kramer
- 👤 Salvatore A. E. Marras
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-02 23:33
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6813; C12Q1/6816; C12Q1/6818
Claims Analysis
Total Claims: 51
Claim 1
Independent Claim
275 words
A fluorescently labeled hairpin-forming oligonucleotide containing: a) a fluorescent emitter moiety having an excitation spectrum and an emission spectrum including a maximum emission wavelength; b) a fluorescent harvester moiety having an excitation spectrum including a maximum excitation wavelength, having an emission spectrum that overlaps the excitation spectrum of the emitter moiety and including a maximum emission wavelength, the emission of the harvester moiety at its maximum emission wavelength having a first magnitude when said harvester moiety is unquenched and stimulated at its maximum excitation wavelength; and c) a quencher moiety capable of quenching the fluorescence of at least one of the emitter moiety and the harvester moiety, said oligonucleotide having a closed conformation including a single-stranded loop and a stem duplex, wherein said quencher moiety is in a quenching relationship to at least one of said harvester and emitter moieties and wherein, when excited at the maximum excitation wavelength of the harvester moiety, emission at the maximum emission wavelength of the harvester moiety is substantially suppressed relative to said first magnitude and emission at the maximum emission wavelength of the emitter moiety has a second magnitude, and said oligonucleotide having an open conformation not including said stem duplex in which said quencher moiety is not in a quenching relationship with said harvester or said emitter moiety wherein, when excited at the maximum excitation wavelength of the harvester moiety, emission at the maximum emission wavelength of the harvester moiety is substantially suppressed relative to said first magnitude, energy is transferred from the harvester moiety to the emitter moiety, and emission at the maximum emission wavelength of the emitter moiety is detectably greater than said second magnitude.
Claim 2
Dependent Claim
275 words
A fluorescently labeled hairpin-forming oligonucleotide containing: a) a fluorescent emitter moiety having an excitation spectrum and an emission spectrum including a maximum emission wavelength; b) a fluorescent harvester moiety having an excitation spectrum including a maximum excitation wavelength, having an emission spectrum that overlaps the excitation spectrum of the emitter moiety and including a maximum emission wavelength, the emission of the harvester moiety at its maximum emission wavelength having a first magnitude when said harvester moiety is unquenched and stimulated at its maximum excitation wavelength; and c) a quencher moiety capable of quenching the fluorescence of at least one of the emitter moiety and the harvester moiety, said oligonucleotide having a closed conformation including a single-stranded loop and a stem duplex, wherein said quencher moiety is in a quenching relationship to at least one of said harvester and emitter moieties and wherein, when excited at the maximum excitation wavelength of the harvester moiety, emission at the maximum emission wavelength of the harvester moiety is substantially suppressed relative to said first magnitude and emission at the maximum emission wavelength of the emitter moiety has a second magnitude, and said oligonucleotide having an open conformation not including said stem duplex in which said quencher moiety is not in a quenching relationship with said harvester or said emitter moiety wherein, when excited at the maximum excitation wavelength of the harvester moiety, emission at the maximum emission wavelength of the harvester moiety is substantially suppressed relative to said first magnitude, energy is transferred from the harvester moiety to the emitter moiety, and emission at the maximum emission wavelength of the emitter moiety is detectably greater than said second magnitude.
Claim 3
Dependent Claim
22 words
The oligonucleotide of claim 1 wherein hybridization of said loop to a target sequence causes said oligonucleotide to assume its open conformation.
Claim 4
Dependent Claim
21 words
The oligonucleotide of claim 2 wherein said quencher moiety is selected from the group consisting of DABCYL and variants of DABCYL.
Claim 5
Dependent Claim
52 words
The oligonucleotide of claim 2 wherein, when said oligonucleotide is in its closed conformation, the emitter moiety is located on said oligonucleotide terminally to said stem duplex, the quencher and harvester moieties are located on said oligonucleotide in said stem duplex, and the harvester and quencher moieties are in a quenching relationship.
Claim 6
Dependent Claim
35 words
The oligonucleotide of claim 2 wherein, when said oligonucleotide is in its open conformation said emission at the maximum emission wavelength of the emitter is at least four times as great as said second magnitude.
Claim 7
Dependent Claim
32 words
The oligonucleotide of claim 2 wherein, when said oligonucleotide is in its closed conformation, said quencher and emitter moieties are located on said oligonucleotide in said stem duplex in a quenching relationship.
Claim 8
Dependent Claim
24 words
The oligonucleotide of claim 2 wherein at least one of said harvester and emitter moieties forms part of the nucleotide chain of said oligonucleotide.
Claim 9
Dependent Claim
25 words
The oligonucleotide of claim 2 wherein at least one of said harvester, emitter and quencher moieties is attached to said oligonucleotide through an alkyl linker.
Claim 10
Dependent Claim
21 words
The oligonucleotide of claim 2 wherein said oligonucleotide includes at least one of the group of modified nucleotides and modified linkages.
Claim 11
Dependent Claim
13 words
The oligonucleotide of claim 9 wherein said oligonucleotide comprises peptide nucleic acid (PNA).
Claim 12
Dependent Claim
10 words
The oligonucleotide of claim 9 wherein said oligonucleotide includes 2'-O-methylribonucleotides.
Claim 13
Dependent Claim
32 words
The oligonucleotide of claim 2 wherein one strand of said stem duplex is complementary to said target strand, whereby said oligonucleotide is capable of serving as a primer for a DNA polymerase.
Claim 14
Dependent Claim
31 words
The oligonucleotide of claim 1 wherein said oligonucleotide includes a terminal extension capable of serving as a priming region for a DNA polymerase when said oligonucleotide is in its closed conformation.
Claim 15
Dependent Claim
30 words
The oligonucleotide of claim 13 wherein said emitter moiety is located on said terminal extension and said quencher and harvester moieties are located on said oligonucleotide in said stem duplex.
Claim 16
Dependent Claim
24 words
The oligonucleotide of claim 13 wherein at least one of said harvester and emitter moieties forms part of the nucleotide chain of said oligonucleotide.
Claim 17
Dependent Claim
25 words
The oligonucleotide of claim 13 wherein at least one of said harvester, emitter and quencher moieties is attached to said oligonucleotide through an alkyl linker.
Claim 18
Dependent Claim
21 words
The oligonucleotide of claim 13 wherein said oligonucleotide includes at least one of the group of modified nucleotides and modified linkages.
Claim 19
Dependent Claim
29 words
A reagent kit comprising ingredients for a nucleic acid amplification, a detector probe that is an oligonucleotide according to claim 2, and instructions for carrying out said amplification reaction.
Claim 20
Dependent Claim
49 words
The kit of claim 18 wherein said amplification is selected from the group consisting of polymerase chain reaction (PCR), strand displacement amplification (SDA), transcription mediated amplification (TMA), ligase chain reaction (LCR), nucleic acid sequence based amplification (NASBA), rolling circle amplification, and amplification of RNA by an RNA-directed RNA polymerase.
Claim 21
Dependent Claim
34 words
The kit according to claim 18 wherein said oligonucleotide is designed to hybridize to a perfectly complementary target sequence but not to hybridize significantly to a sequence differing therefrom by at least one nucleotide.
Claim 22
Dependent Claim
35 words
The kit according to claim 18 wherein the emitter moiety is located on said oligonucleotide terminally to said stem duplex and the quencher and harvester moieties are located on said oligonucleotide in said stem duplex.
Claim 23
Dependent Claim
41 words
A reagent kit for an amplification reaction that includes at least one primer, comprising the ingredients for said amplification assay and instructions for carrying out said amplification assay, wherein said at least one primer is an oligonucleotide according to claim 13.
Claim 24
Dependent Claim
52 words
The kit according to claim 22 wherein said amplification reaction is selected from the group consisting of the polymerase chain reaction (PCR), strand displacement amplification (SDA), transcription mediated amplification (TMA), ligase chain reaction (LCR), nucleic acid sequence based amplification (NASBA), rolling circle amplification, and amplification of RNA by an RNA-directed RNA polymerase.
Claim 25
Dependent Claim
11 words
The kit according to claim 23 wherein said oligonucleotide includes 2'-O-methylribonucleotides.
Claim 26
Dependent Claim
39 words
A detection assay comprising adding to a sample that might contain a target strand at least one detector probe that is an oligonucleotide according to claim 2 and detecting fluorescence emission from said at least one probe's emitter moiety.
Claim 27
Dependent Claim
19 words
The assay according to claim 25 further comprising detecting fluorescence emission from said at least one probe's harvester moiety.
Claim 28
Dependent Claim
35 words
The assay according to claim 26 further comprising adding said at least one detector probe to a sample that does not contain said target strand and detecting fluorescence emission of said harvester and emitter moieties.
Claim 29
Dependent Claim
34 words
The assay according to claim 25 further comprising adding said detector probe to a sample that does not contain said target strand and detecting fluorescence emission of said at least one probe's emitter moiety.
Claim 30
Dependent Claim
41 words
The assay according to claim 25 wherein the emitter moiety is of said at least one probe located on said oligonucleotide terminally to said stem duplex and the quencher and harvester moieties are located on said oligonucleotide in said stem duplex.
Claim 31
Dependent Claim
33 words
The assay according to claim 25 wherein one of the harvester and emitter moieties of said at least one probe is a phosphoramidite derivative of a fluorophore that is incorporated in said oligonucleotide.
Claim 32
Dependent Claim
23 words
The assay according to claim 25 wherein said at least one probe comprises at least two probes, each having a different emitter moiety.
Claim 33
Dependent Claim
22 words
The assay according to claim 25, wherein said assay is selected from the group consisting of in situ and in vivo assays.
Claim 34
Dependent Claim
84 words
An amplification assay comprising adding to a sample that might contain a target strand the reagents to perform an amplification reaction selected from the group consisting of the polymerase chain reaction (PCR), strand displacement amplification (SDA), transcription mediated amplification (TMA), ligase chain reaction (LCR), nucleic acid sequence based amplification (NASBA), rolling circle amplification, and amplification of RNA by an RNA-directed RNA polymerase, and at least one detector probe according to claim 2 and detecting fluorescence emission from said at least one probe's emitter moiety.
Claim 35
Dependent Claim
22 words
The assay according to claim 33, wherein said assay is selected from the group consisting of in situ and in vivo assays.
Claim 36
Dependent Claim
19 words
The assay according to claim 33 further comprising detecting fluorescence emission from said at least one probe's harvester moiety.
Claim 37
Dependent Claim
39 words
The assay according to claim 35 further comprising adding said at least one detector probe to a sample that does not contain said target strand and detecting fluorescence emission of said at least one probe's harvester and emitter moieties.
Claim 38
Dependent Claim
37 words
The assay according to claim 33 further comprising adding said at least one detector probe to a sample that does not contain said target strand and detecting fluorescence emission of said at least one probe's emitter moiety.
Claim 39
Dependent Claim
35 words
The assay according to claim 33 wherein the emitter moiety is located on said oligonucleotide terminally to said stem duplex and the quencher and harvester moieties are located on said oligonucleotide in said stem duplex.
Claim 40
Dependent Claim
33 words
The assay according to claim 33 wherein one of the harvester and emitter moieties of said at least one probe is a phosphoramidite derivative of a fluorophore that is incorporated in said oligonucleotide.
Claim 41
Dependent Claim
23 words
The assay according to claim 33 wherein said at least one probe comprises at least two probes, each having a different emitter moiety.
Claim 42
Dependent Claim
11 words
The assay according to claim 33 wherein said oligoncleotide includes 2'-O-methylribonucleotides.
Claim 43
Dependent Claim
52 words
An amplification assay comprising an amplification reaction that includes at least one primer comprising the steps of adding to a sample that might contain a target strand the reagents to perform said amplification reaction, said reagents including at least one primer according to claim 12, and detecting fluorescence of said emitter moiety.
Claim 44
Dependent Claim
22 words
The assay according to claim 42, wherein said assay is selected from the group consisting of in situ and in vivo assays.
Claim 45
Dependent Claim
22 words
The amplification assay according to claim 42 wherein said at least one primer according to claim 12 comprises at least two primers.
Claim 46
Dependent Claim
52 words
An amplification assay comprising an amplification reaction that includes at least one primer comprising the steps of adding to a sample that might contain a target strand the reagents to perform said amplification reaction, said reagents including at least one primer according to claim 13, and detecting fluorescence of said emitter moiety.
Claim 47
Dependent Claim
22 words
The amplification assay according to claim 45 wherein said at least one primer according to claim 13 comprises at least two primers.
Claim 48
Dependent Claim
16 words
The amplification assay according to claim 45 further comprising detecting fluorescence emission from said harvester moiety.
Claim 49
Dependent Claim
36 words
The amplification assay according to claim 45 wherein the emitter moiety is located on said oligonucleotide terminally to said stem duplex and the quencher and harvester moieties are located on said oligonucleotide in said stem duplex.
Claim 50
Dependent Claim
33 words
The assay according to claim 45 wherein one of the harvester and emitter moieties of said at least one probe is a phosphoramidite derivative of a fluorophore that is incorporated in said oligonucleotide.
Claim 51
Dependent Claim
23 words
The assay according to claim 45 wherein said at least one probe comprises at least two probes, each having a different emitter moiety.
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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) | 2003-03-14 | 2003-09-13 | 2004-03-14 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2007-03-14 | 2007-09-13 | 2008-03-14 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2011-03-14 | 2011-09-13 | 2012-03-14 | $7700.00 | Window Closed / Lapsed |