US5445935A
Expired / Lapsed
US
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Quantitative detection of macromolecules with fluorescent oligonucleotides
Assignee: R-P TECHNOLOGIES
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Filing: 1992-11-23
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Granted: 1995-08-29
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Expires: 2012-11-23
Abstract
Abstract A method is described by which the association between an oligonucleotide labeled by attachment of a fluorophore and another macromolecule such as a protein or nucleic acid may be determined quantitatively in solution accurately and with high sensitivity. In the performance of this method the polarization of fluorescence of an extrinsic fluorescence probe that is covalently coupled to the oligonucleotide is determined. Changes in fluorescence polarization are related directly to the degree of association between the labeled oligonucleotide and another macromolecule and may be used to quantify the association. Because of its high sensitivity and accuracy, this method may be used to make reliable quantitative measurements of very small amounts of complexes formed between labeled oligonucleotides and proteins, nucleic acids or other macromolecules. The method also allows the accurate calculation of important biochemical parameters such as dissociation constants. The method, which is rapid and solution-based, has a broad range of applications in biochemistry, genetic cloning and molecular biology, as well as in clinical diagnostics.
Inventors
- 👤 Catherine A. Royer
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-26 08:55
CPC Classifications
G; G01; G01N; G01N21/00; G01N21/62; G01N21/63; G01N21/64; G01N21/645; C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6813; C12Q1/6816; G01N21/6428; G01N21/6445; C12Q2600/00; C12Q2600/156; G01N2021/6463
Claims Analysis
Total Claims: 13
Claim 1
Independent Claim
190 words
A method for measuring the binding of a polynucleotide to a macromolecule to form a complex which comprises: i) labelling said polynucleotide with a fluorescent label; ii) obtaining a first measurement of the polarization of the fluorescent emission from said label; iii) contacting said labelled polynucleotide with said macromolecule, under conditions such that the concentration of said labelled polynucleotide ranges from 10 picomolar to 1 nanomolar; iv) obtaining a second measurement of the polarization of the fluorescent emission from said label; v) comparing said first measurement with said second measurement; and vi) detecting formation of said complex by observing an increase in the polarization measured in step (iv) compared to the polarization measured in step (ii); wherein said measurements of fluorescence polarization are made using an apparatus having a component selected from the group consisting of an apparatus having a Xenon arc lamp light source and a UV grade film polarizer, an apparatus having a Xenon arc lamp light source coupled by a fiber optic cable to a sample compartment and a UV grade film polarizer mounted directly on the sample compartment, and an apparatus having a laser light source.
Claim 2
Independent Claim
190 words
A method for measuring the binding of a polynucleotide to a macromolecule to form a complex which comprises: i) labelling said polynucleotide with a fluorescent label; ii) obtaining a first measurement of the polarization of the fluorescent emission from said label; iii) contacting said labelled polynucleotide with said macromolecule, under conditions such that the concentration of said labelled polynucleotide ranges from 10 picomolar to 1 nanomolar; iv) obtaining a second measurement of the polarization of the fluorescent emission from said label; v) comparing said first measurement with said second measurement; and vi) detecting formation of said complex by observing an increase in the polarization measured in step (iv) compared to the polarization measured in step (ii); wherein said measurements of fluorescence polarization are made using an apparatus having a component selected from the group consisting of an apparatus having a Xenon arc lamp light source and a UV grade film polarizer, an apparatus having a Xenon arc lamp light source coupled by a fiber optic cable to a sample compartment and a UV grade film polarizer mounted directly on the sample compartment, and an apparatus having a laser light source.
Claim 3
Dependent Claim
12 words
A method as recited in claim 1, wherein said label is fluorescein.
Claim 4
Dependent Claim
23 words
A method as recited in claim 1, wherein said polynucleotide is a single-stranded oligodeoxyribonucleotide of length less than or equal to 40 bases.
Claim 5
Dependent Claim
14 words
A method as recited in claim 3, wherein said macromolecule is a second polynucleotide.
Claim 6
Dependent Claim
23 words
A method as recited in claim 1, wherein said polynucleotide is a double-stranded oligodeoxyribonucleotide of length less than or equal to 40 basepairs.
Claim 7
Dependent Claim
13 words
A method as recited in claim 1, wherein said macromolecule is a protein.
Claim 8
Dependent Claim
22 words
A method as recited in claim 1, wherein said macromolecule is present at a concentration ranging from 10 picomolar to 1 nanomolar.
Claim 9
Dependent Claim
93 words
The method of claim 7, which further comprises the following steps: vii) contacting unlabeled polynucleotide at a concentration of 10 picomolar to 1 nanomolar with the labelled polynucleotide and macromolecule of step (iii), said unlabeled polynucleotide having the same nucleotide sequence as said labelled polynucleotide; viii) obtaining a third measurement of the polarization of the fluorescent emission from said fluorescently-labelled oligonucleotide ix) comparing said third measurement to said first and second measurements, wherein the binding is measured by observing a change in the polarization measured in each of steps (ii), (iv) and (viii).
Claim 10
Independent Claim
202 words
A method for detecting the presence of wild-type or mutant gene in a patient which comprises: i) obtaining a sample of DNA from the patient; ii) obtaining a first measurement of the polarization of a fluorescent emission from a fluorescently-labelled oligonucleotide probe which is capable of binding exclusively to either wild-type sample DNA or mutant DNA; iii) contacting said probe at a concentration of 10 picomolar to 1 nanomolar under conditions such that the probe binds exclusively to either wild-type DNA or mutant DNA; iv) obtaining a second measurement of the polarization of the fluorescent emission from said fluorescently-labelled oligonucleotide; and v) comparing said second measurement to said first measurement; wherein a change in polarization measured in step (ii) and step (iv) indicates the presence of the wild-type or mutant gene; wherein said measurements of fluorescence polarization are made using an apparatus selected from the group consisting of an apparatus having a Xenon arc lamp light source and a UV grade film polarizer, an apparatus having a Xenon arc lamp light source coupled by a fiber optic cable to a sample compartment and a UV grade film polarizer mounted directly on the sample compartment, and an apparatus having a laser light source.
Claim 11
Independent Claim
202 words
A method for detecting the presence of wild-type or mutant gene in a patient which comprises: i) obtaining a sample of DNA from the patient; ii) obtaining a first measurement of the polarization of a fluorescent emission from a fluorescently-labelled oligonucleotide probe which is capable of binding exclusively to either wild-type sample DNA or mutant DNA; iii) contacting said probe at a concentration of 10 picomolar to 1 nanomolar under conditions such that the probe binds exclusively to either wild-type DNA or mutant DNA; iv) obtaining a second measurement of the polarization of the fluorescent emission from said fluorescently-labelled oligonucleotide; and v) comparing said second measurement to said first measurement; wherein a change in polarization measured in step (ii) and step (iv) indicates the presence of the wild-type or mutant gene; wherein said measurements of fluorescence polarization are made using an apparatus selected from the group consisting of an apparatus having a Xenon arc lamp light source and a UV grade film polarizer, an apparatus having a Xenon arc lamp light source coupled by a fiber optic cable to a sample compartment and a UV grade film polarizer mounted directly on the sample compartment, and an apparatus having a laser light source.
Claim 12
Independent Claim
203 words
A method for determining the amount of a DNA binding protein which comprises: i) fluorescently labelling an oligonucleotide having a nucleotide sequence recognized by a DNA-binding protein, wherein said oligonucleotide may be either double-stranded or single stranded; ii) obtaining a sample containing the DNA-binding protein to be tested; iii) obtaining a first measurement of the polarization of the fluorescent emission from said fluorescently-labelled oligonucleotide; iv) contacting said fluorescently-labelled oligonucleotide with said sample under conditions such that the concentration of said fluorescently-labelled polynucleotide ranges from 10 picamolar to 1 nanomolar; v) obtaining a second measurement of the polarization of the fluorescent emission from said fluorescently-labelled oligonucleotide; vi) comparing said second measurement to said first measurement; wherein the amount of change between the first and second measurements determines the amount of DNA binding protein, wherein said measurements of fluorescence polarization are made using an apparatus selected from the group consisting of an apparatus having a Xenon arc lamp light source and a UV grade film polarizer an apparatus having a Xenon arc lamp light source coupled by a fiber optic cable to a sample compartment and a UV grade film polarizer mounted directly on the sample compartment, and an apparatus having a laser light source.
Claim 13
Independent Claim
203 words
A method for determining the amount of a DNA binding protein which comprises: i) fluorescently labelling an oligonucleotide having a nucleotide sequence recognized by a DNA-binding protein, wherein said oligonucleotide may be either double-stranded or single stranded; ii) obtaining a sample containing the DNA-binding protein to be tested; iii) obtaining a first measurement of the polarization of the fluorescent emission from said fluorescently-labelled oligonucleotide; iv) contacting said fluorescently-labelled oligonucleotide with said sample under conditions such that the concentration of said fluorescently-labelled polynucleotide ranges from 10 picamolar to 1 nanomolar; v) obtaining a second measurement of the polarization of the fluorescent emission from said fluorescently-labelled oligonucleotide; vi) comparing said second measurement to said first measurement; wherein the amount of change between the first and second measurements determines the amount of DNA binding protein, wherein said measurements of fluorescence polarization are made using an apparatus selected from the group consisting of an apparatus having a Xenon arc lamp light source and a UV grade film polarizer an apparatus having a Xenon arc lamp light source coupled by a fiber optic cable to a sample compartment and a UV grade film polarizer mounted directly on the sample compartment, and an apparatus having a laser light source.
References Cited (Backward Citations)
US4269511A
Cited
US5066426A
Cited
US4585862A
Cited
US4668640A
Cited
US4952691A
Cited
EP0194472A2
Cited
US4902630A
Cited
US4784961A
Cited
EP0382433A2
Cited
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