US6258569B1
Expired / Lapsed
US
⏱️ 0.0 years remaining
Hybridization assay using self-quenching fluorescence probe
Assignee: Applied Biosystems LLC
•
Filing: 1999-11-08
•
Granted: 2001-07-10
•
Expires: 2019-11-08
Abstract
Abstract Provided is a method of nucleic acid amplification. In one embodiment, the method comprises performing nucleic acid amplification on a target polynucleotide using a nucleic acid polymerase having 5' to 3' nuclease activity, a primer capable of hybridizing to the target polynucleotide, and an oligonucleotide probe under amplification conditions such that the probe hybridizes to the target polynucleotide 3' relative to the primer and the probe does not hybridize with itself to form a hairpin structure. The oligonucleotide probe has at one end a fluorescent reporter and at the other end a quencher that quenches the fluorescence of the reporter molecule when both the fluorescent reporter and quencher are attached to the probe. Digestion of the oligonucleotide probe by the polymerase during amplification is effective to separate the reporter from the quencher, whereby a fluorescence signal of the reporter is increased.
Inventors
- 👤 Kenneth J. Livak
- 👤 Susan J. A. Flood
- 👤 Jeffrey Mamoro
- 👤 Khairuzzaman Bashar Mullah
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-31 00:51
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6813; C12Q1/6816; C12Q1/6818; C12Q1/6834; C12Q1/6837
Claims Analysis
Total Claims: 37
Claim 1
Independent Claim
138 words
A method of nucleic acid amplification comprising: performing nucleic acid amplification on a target polynucleotide using a nucleic acid polymerase having 5′ to 3′ nuclease activity, a primer capable of hybridizing to the target polynucleotide, and an oligonucleotide probe under amplification conditions such that the probe hybridizes to the target polynucleotide 3′ relative to the primer and the probe does not hybridize with itself to form a hairpin structure, the oligonucleotide probe having at one end a fluorescent reporter and at the other end a quencher that quenches the fluorescence of the reporter molecule when both the fluorescent reporter and quencher are attached to the probe, under conditions such that digestion of the oligonucleotide probe by the polymerase during amplification is effective to separate the reporter from the quencher, whereby a fluorescence signal of the reporter is increased.
Claim 2
Independent Claim
138 words
A method of nucleic acid amplification comprising: performing nucleic acid amplification on a target polynucleotide using a nucleic acid polymerase having 5′ to 3′ nuclease activity, a primer capable of hybridizing to the target polynucleotide, and an oligonucleotide probe under amplification conditions such that the probe hybridizes to the target polynucleotide 3′ relative to the primer and the probe does not hybridize with itself to form a hairpin structure, the oligonucleotide probe having at one end a fluorescent reporter and at the other end a quencher that quenches the fluorescence of the reporter molecule when both the fluorescent reporter and quencher are attached to the probe, under conditions such that digestion of the oligonucleotide probe by the polymerase during amplification is effective to separate the reporter from the quencher, whereby a fluorescence signal of the reporter is increased.
Claim 3
Dependent Claim
15 words
The method of claim 1 , wherein the polymerase is a thermostable nucleic acid polymerase.
Claim 4
Dependent Claim
20 words
The method of claim 1 , wherein the reporter is a fluorescein dye and the quencher is a rhodamine dye.
Claim 5
Dependent Claim
19 words
The method of claim 3 , wherein the fluorescent reporter is attached to the 5′ end of the probe.
Claim 6
Dependent Claim
16 words
The method of claim 4 , wherein the probe is 15 to 60 nucleotides in length.
Claim 7
Dependent Claim
16 words
The method of claim 4 , wherein the probe is 18 to 30 nucleotides in length.
Claim 8
Dependent Claim
19 words
The method of claim 3 , wherein the fluorescent reporter is attached to the 3′ end of the probe.
Claim 9
Dependent Claim
16 words
The method of claim 7 , wherein the probe is 15 to 60 nucleotides in length.
Claim 10
Dependent Claim
16 words
The method of claim 7 , wherein the probe is 18 to 30 nucleotides in length.
Claim 11
Dependent Claim
19 words
The method of claim 1 , wherein the fluorescent reporter is attached to the 5′ end of the probe.
Claim 12
Dependent Claim
16 words
The method of claim 10 , wherein the probe is 15 to 60 nucleotides in length.
Claim 13
Dependent Claim
16 words
The method of claim 10 , wherein the probe is 18 to 30 nucleotides in length.
Claim 14
Dependent Claim
19 words
The method of claim 1 , wherein the fluorescent reporter is attached to the 3′ end of the probe.
Claim 15
Dependent Claim
16 words
The method of claim 13 , wherein the probe is 15 to 60 nucleotides in length.
Claim 16
Dependent Claim
16 words
The method of claim 13 , wherein the probe is 18 to 30 nucleotides in length.
Claim 17
Dependent Claim
16 words
The metod of claim 1 , wherein the repoter or the quencher comprises a rhodamine dye.
Claim 18
Dependent Claim
16 words
The method of claim 1 , wherein the reporter or the quencher comprises a fluorescein dye.
Claim 19
Dependent Claim
25 words
The method of claim 1 , wberein the reporter or quencher is selected from the group consisting of a xanthene dye and a naphthylamine dye.
Claim 20
Dependent Claim
20 words
The method of claim 1 , wherein the oligonucleotide probe is not capable of extension by the nucleic acid polymerase.
Claim 21
Dependent Claim
15 words
The method of claim 19 , wherein the polymerase is a thermostable nucleic acid polymerase.
Claim 22
Dependent Claim
20 words
The method of claim 19 , wherein the reporter is a fluorescein dye and the quencher is a rhodamine dye.
Claim 23
Dependent Claim
19 words
The method of claim 21 , wherein the fluorescent reporter is attached to the 5′ end of the probe.
Claim 24
Dependent Claim
16 words
The method of claim 22 , wherein the probe is 15 to 60 nucleotides in length.
Claim 25
Dependent Claim
16 words
The method of claim 22 , wherein the probe is 18 to 30 nucleotides in length.
Claim 26
Dependent Claim
19 words
The method of claim 21 , wherein the fluorescent reporter is attached to the 3′ end of the probe.
Claim 27
Dependent Claim
16 words
The method of claim 25 , wherein the probe is 15 to 60 nucleotides in length.
Claim 28
Dependent Claim
16 words
The method of claim 25 , wherein the probe is 18 to 30 nucleotides in length.
Claim 29
Dependent Claim
19 words
The method of claim 19 , wherein the fluorescent reporter is attached to the 5′ end of the probe.
Claim 30
Dependent Claim
16 words
The method of claim 28 , wherein the probe is 15 to 60 nucleotides in length.
Claim 31
Dependent Claim
16 words
The method of claim 28 , wherein the probe is 18 to 30 nucleotides in length.
Claim 32
Dependent Claim
19 words
The method of claim 19 , wherein the fluorescent reporter is attached to the 3′ end of tbe probe.
Claim 33
Dependent Claim
16 words
The method of claim 31 , wherein the probe is 15 to 60 nucleotides in length.
Claim 34
Dependent Claim
16 words
The method of claim 31 , wherein the probe is 18 to 30 nucleotides in length.
Claim 35
Dependent Claim
17 words
The method of claim 19 , wherein the fluorescent reporter or the quencher comprises a rhodamine dye.
Claim 36
Dependent Claim
17 words
The method of claim 19 , wherein the fluorescent reporter or the quencher comprises a fluorescein dye.
Claim 37
Dependent Claim
25 words
The method of claim 19 , wherei dte reporter or quencher is selected from the group consisting of a xanthene dye and a naphthylamine dye.
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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) | 2004-07-09 | 2005-01-08 | 2005-07-10 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2008-07-09 | 2009-01-08 | 2009-07-10 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2012-07-09 | 2013-01-08 | 2013-07-10 | $7700.00 | Window Closed / Lapsed |