US5736333A
Expired / Lapsed
US
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Passive internal references for the detection of nucleic acid amplification products
Assignee: Applied Biosystems LLC
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Filing: 1996-06-04
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Granted: 1998-04-07
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Expires: 2016-06-04
Abstract
Abstract The invention relates to passive internal references for use in quantitating the formation of amplification products in a nucleic amplification reaction. The internal amplification reference molecules of the invention comprise a first and second fluorophore joined together through a backbone connector. The first and second fluorophores are joined on the backbone in a configuration that permits the energy transfer from the first fluorophore to the second fluorophore. The backbone connector is selected so as not to bind to the target nucleic acid sequence under nucleic acid amplification conditions. Preferably, the backbone connector is a polynucleotide. Another aspect of the invention is to provide passive internal reference molecule containing reagent compositions for use in nucleic acid amplification reactions. The compositions comprise the internal amplification reference molecule of the invention and a nucleic acid amplification reaction buffer. The reagent compositions, optionally, include additional components required for nucleic acid amplification reactions. The invention also provides improved methods of measuring the amount of amplification product in nucleic acid amplification reactions employing fluorescer-quencher probe assays, including methods for the real-time measurement of amplification product formation. The methods comprise the step of adding the internal reference molecule of the invention to the amplification reaction mixture. Fluorescence of the second fluorophore on the internal reference may then be measured and used to calculate changes in fluorescence of the fluorophore on a fluorescer-quencher probe.
Inventors
- 👤 Kenneth J. Livak
- 👤 Lincoln J. McBride
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-05 10:26
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6844; C12Q1/6851
Claims Analysis
Total Claims: 23
Claim 1
Independent Claim
76 words
A reagent composition, said composition comprising: a nucleic acid amplification buffer, and an internal reference molecule, said internal reference molecule comprising a first fluorophore, a second fluorophore, and a backbone connector that does not hybridize in a sequence specific manner to a polynucleotide for amplification under nucleic acid amplification conditions, wherein the backbone connector joins the first and second fluorophores so as to permit the transfer of energy from the first fluorophore to the second fluorophore.
Claim 2
Dependent Claim
76 words
A reagent composition, said composition comprising: a nucleic acid amplification buffer, and an internal reference molecule, said internal reference molecule comprising a first fluorophore, a second fluorophore, and a backbone connector that does not hybridize in a sequence specific manner to a polynucleotide for amplification under nucleic acid amplification conditions, wherein the backbone connector joins the first and second fluorophores so as to permit the transfer of energy from the first fluorophore to the second fluorophore.
Claim 3
Dependent Claim
13 words
A composition according to claim 1, wherein the backbone connector is a polynucleotide.
Claim 4
Dependent Claim
16 words
A composition according to claim 2, wherein the polynucleotide is 2 to 25 nucleotides in length.
Claim 5
Dependent Claim
11 words
A composition according to claim 3, wherein the polynucleotide is (dT)8.
Claim 6
Dependent Claim
27 words
A composition according to claim 1 wherein the first fluorophore is selected from the group consisting of fluorescein, 6-carboxyfluorescein, 2',4',5',7',-tetrachloro-4,7-dichlorofluorescein, 2',7'-dimethoxy-4',5'-6-carboxyrhodamine (JOE), N',N',N',N'-tetramethyl-6-carboxyrhodamine (TAMRA) and 6-carboxy-X-rhodamine (ROX).
Claim 7
Dependent Claim
12 words
A composition according to claim 5, wherein the first fluorophore is 6-carboxyfluorescein.
Claim 8
Dependent Claim
27 words
A composition according to claim 5 wherein the second fluorophore is selected from the group consisting of fluorescein, 6-carboxyfluorescein, 2',4',5',7',-tetrachloro-4,7-dichlorofluorescein, 2',7'-dimethoxy-4',5'-6-carboxyrhodamine (JOE), N',N',N',N'-tetramethyl-6-carboxyrhodamine (TAMRA) and 6-carboxy-X-rhodamine (ROX).
Claim 9
Dependent Claim
12 words
A composition according to claim 6, wherein the second fluorophore is 6-carboxy-X-rhodamine.
Claim 10
Dependent Claim
90 words
In a method of measuring the amount of an amplification product of a polynucleotide for amplification in a nucleic acid reaction wherein the improvement comprises, adding an internal amplification reference molecule to the amplification reaction, said internal reference molecule comprising a first fluorophore, a second fluorophore, and a backbone connector that does not hybridize to the polynucleotide for amplification under nucleic acid amplification conditions, wherein the backbone connector joins the first and second fluorophores so as to permit the transfer of energy from the first fluorophore to the second fluorophore.
Claim 11
Dependent Claim
90 words
In a method of measuring the amount of an amplification product of a polynucleotide for amplification in a nucleic acid reaction wherein the improvement comprises, adding an internal amplification reference molecule to the amplification reaction, said internal reference molecule comprising a first fluorophore, a second fluorophore, and a backbone connector that does not hybridize to the polynucleotide for amplification under nucleic acid amplification conditions, wherein the backbone connector joins the first and second fluorophores so as to permit the transfer of energy from the first fluorophore to the second fluorophore.
Claim 12
Dependent Claim
16 words
A method according to claim 9, wherein the nucleic acid amplification reaction is measured in real-time.
Claim 13
Dependent Claim
13 words
A method according to claim 9, wherein the backbone connector is a polynucleotide.
Claim 14
Dependent Claim
16 words
A method according to claim 10, wherein the polynucleotide is 2 to 25 nucleotides in length.
Claim 15
Dependent Claim
11 words
A method according to claim 12, wherein the polynucleotide is (dT)8.
Claim 16
Dependent Claim
27 words
A method according to claim 9 wherein the first fluorophore is selected from the group consisting of fluorescein, 6-carboxyfluorescein, 2',4',5',7',-tetrachloro-4,7-dichlorofluorescein, 2',7'-dimethoxy-4',5'-6-carboxyrhodamine (JOE), N',N',N',N'-tetramethyl-6-carboxyrhodamine (TAMRA) and 6-carboxy-X-rhodamine (ROX).
Claim 17
Dependent Claim
12 words
A method according to claim 14, wherein the first fluorophore is 6-carboxyfluorescein.
Claim 18
Dependent Claim
27 words
A method according to claim 14 wherein the second fluorophore is selected from the group consisting of fluorescein, 6-carboxyfluorescein, 2',4',5',7',-tetrachloro-4,7-dichlorofluorescein, 2',7'-dimethoxy-4',5'-6-carboxyrhodamine (JOE), N',N',N',N'-tetramethyl-6-carboxyrhodamine (TAMRA) and 6-carboxy-X-rhodamine (ROX).
Claim 19
Dependent Claim
12 words
A method according to claim 15, wherein the second fluorophore is 6-carboxy-X-rhodamine.
Claim 20
Dependent Claim
37 words
A kit for performing nucleic acid amplification reactions, said kit comprising a reagent composition according to claim 1 and one or more reagents selected from the group consisting of a fluorescer-quencher probe and a thermostable DNA polymerase.
Claim 21
Dependent Claim
13 words
A kit according to claim 18, wherein said reagent is a fluorescer-quencher probe.
Claim 22
Dependent Claim
14 words
A kit according to claim 18, wherein said reagent is a thermostable DNA polymerase.
Claim 23
Dependent Claim
13 words
A kit according to claim 20, said kit further comprising a fluorescer-quencher probe.
References Cited (Backward Citations)
US5210015A
Cited
US5527684A
Cited
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
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| 3.5 Years (Year 4) | 2001-04-06 | 2001-10-06 | 2002-04-07 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2005-04-06 | 2005-10-06 | 2006-04-07 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2009-04-06 | 2009-10-06 | 2010-04-07 | $7700.00 | Window Closed / Lapsed |