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US 7,452,668 B2Granted
Electrophoretic analysis of molecules using immobilized probes
AssigneeExact Sciences Corporation
InventorsTruett C. Boles, Andrew R. Muir, Stephen J. Kron, Ezra S. Abrams
Life of this patent
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Abstract
Methods of detecting target molecules using electrophoresis and media containing immobilized capture are described.
Claims73
A method of detecting the presence or absence of one or more target molecules in a test sample, wherein the one or more target molecules has a defined mobility in an appropriate electrophoretic medium when the medium is exposed to an electric field and the target molecule can form binding complexes with capture probes, comprising the steps of:
- a) copolymerizing one or more capture probes with a material capable of forming a matrix suitable for electrophoresis to form an electrophoretic medium containing immobilized capture probes covalently attached to the electrophoretic medium, wherein said one or more capture probes are modified with a 5′-acrylamide moiety and are selected from the group consisting of a nucleic acid, modified nucleic acid and a nucleic acid analog, and wherein said one or more capture probes are immobilized in a two dimensional array within the electrophoretic medium;
- b) introducing a test sample into the electrophoretic medium;
- c) subjecting the electrophoretic medium to an electric field resulting in the electrophoretic migration of the test sample into the region or regions of the electrophoretic medium containing immobilized capture probes, under conditions suitable for the target molecules to bind to the immobilized probes;
- and
- d) detecting the presence, or absence of target molecules or target molecule/capture probe complexes immobilized in the medium,
- thereby detecting the presence or absence of one or more target molecules in the test sample.
3 more elements
A method for purifying or concentrating target molecules from a complex test sample, comprising:
- a) copolymerizing one or more capture probes modified with a 5′-acrylamide moiety and selected from the group consisting of a nucleic acid, modified nucleic acid and a nucleic acid analog with a material capable of forming a matrix suitable for electrophoresis to form an electrophoretic medium containing immobilized capture probes covalently attached to the electrophoretic medium, wherein said one or more capture probes are immobilized in a two dimensional array within the electrophoretic medium;
- b) introducing a test sample into the electrophoretic medium;
- c) subjecting the electrophoretic medium to an electric field resulting in the electrophoretic migration of the test sample through the medium, under conditions and time sufficient for the target molecules in the test sample to specifically bind to the capture probes, thereby forming target molecule/capture probe complexes, and for non-target molecules to migrate through and elute from the medium, wherein only target molecules bind to the capture probes and are immobilized in the medium;
- d) treating the electrophoretic medium to release at least one of the following:
- i) the one or more capture probes,
- ii) the target molecules, or
- iii) the target/capture probe binding complexes;
- and
- e) eluting the target molecules or target/capture probe complexes from the medium, whereby the released and eluted target molecules are substantially pure.
6 more elements
A method of detecting one or more target molecules in a test sample, comprising the steps of:
- a) copolymerizing one or more nucleic acid aptamer capture probes modified with a 5′-acrylamide moiety with a material capable of forming a matrix suitable for electrophoresis to form an electrophoretic medium containing at least one immobilized nucleic acid aptamer capture probe covalently attached to the electrophoretic medium, wherein the aptamer capture probe is a thrombin-binding DNA aptamer;
- b) introducing a test sample into the electrophoretic medium;
- c) subjecting the electrophoretic medium to an electric field resulting in the electrophoretic migration of the test sample into the electrophoretic medium under conditions suitable for the target molecules to bind to the at least one immobilized aptamer capture probe, wherein the target molecule is human thrombin and is SEQ ID No.1;
- and
- d) detecting the presence or absence of target molecule/aptamer capture probe complexes immobilized in the medium,
- thereby detecting the presence or absence of one or more target molecules in the test sample.
3 more elements
A method of detecting one or more target molecules in a test sample, comprising the steps of:
- a) copolymerizing one or more nucleic acid aptamer capture probes modified with a 5′-acrylamide moiety with a material capable of forming a matrix suitable for electrophoresis to form an electrophoretic medium containing at least one immobilized nucleic acid aptamer capture probe covalently attached to the electrophoretic medium;
- b) introducing a test sample into the electrophoretic medium:
- c) subjecting the electrophoretic medium to an electric field resulting in the electrophoretic migration of the test sample into the electrophoretic medium under conditions suitable for the target molecules to bind to the at least one immobilized aptamer capture probe, wherein the target molecule is selected from the group consisting of:
- SEQ ID No.2 and SEQ ID No.3;
- and
- d) detecting the presence or absence of target molecule/aptamer capture probe complexes immobilized in the medium,
- thereby detecting the presence or absence of one or more target molecules in the test sample.
4 more elements
A method for purifying molecules in a complex test sample comprising target molecules and non-target molecules, comprising:
- a) copolymerizing one or more capture probes modified with a 5′-acrylamide moiety and selected from the group consisting of a nucleic acid, modified nucleic acid and a nucleic acid analog with a material capable of forming a matrix suitable for electrophoresis to form an electrophoretic medium containing immobilized capture probes covalently attached to the electrophoretic medium, wherein said one or more capture probes are immobilized in a two dimensional array within the electrophoretic medium;
- b) introducing a test sample into the electrophoretic medium;
- c) subjecting the electrophoretic medium to an electric field resulting in the electrophoretic migration of the test sample through the medium, under conditions and time sufficient for the target molecules in the test sample to specifically bind to the capture probes, thereby forming target molecule/capture probe complexes and allowing non-target molecules to migrate through and elute from the medium, wherein only target molecules bind to the capture probes and are immobilized in the medium,
- thereby subtracting out the target molecule from the sample preparation and purifying molecules in the complex test sample.
1 more element
A method of detecting the presence or absence of one or more target molecules in a test sample, wherein the one or more target molecules have a defined mobility in an appropriate electrophoretic medium when the medium is exposed to an electric field and the one or more target molecules can form binding complexes with capture probes, comprising the steps of:
- a) copolymerizing one or more capture probes with a material capable of forming a matrix suitable for electrophoresis, wherein said copolymerization forms an electrophoretic medium containing immobilized capture probes covalently attached to the electrophoretic medium, and wherein said one or more capture probes are modified with a monomer capable of forming a matrix suitable for electrophoresis, and wherein said capture probes are selected from the group consisting of a nucleic acid, modified nucleic acid and a nucleic acid analog, and wherein one or more capture probes are immobilized in a two dimensional array within the electrophoretic medium,
- b) introducing a test sample into the electrophoretic medium;
- c) subjecting the electrophoretic medium to an electric field resulting in the electrophoretic migration of the test sample into the region or regions of the electrophoretic medium containing immobilized capture probes, under conditions suitable for the target molecules to bind to the immobilized probes;
- and
- d) detecting the presence or absence of target molecules or target molecule/capture probe complexes immobilized in the medium,
- thereby detecting the presence or absence of one or more target molecules in the test sample.
3 more elements
A method for purifying or concentrating target molecules from a complex test sample, comprising:
- a) copolymerizing one or more capture probes with a material capable of forming a matrix suitable for electrophoresis, wherein said copolymerization forms an electrophoretic medium containing immobilized capture probes covalently attached to the electrophoretic medium, and wherein said one or more capture probes are modified with a monomer capable of forming a matrix suitable for electrophoresis, and wherein said capture probes are selected from the group consisting of a nucleic acid, modified nucleic acid and a nucleic acid analog, and wherein one or more capture probes are immobilized in a two dimensional array within the electrophoretic medium,
- b) introducing a test sample into the electrophoretic medium;
- c) subjecting the electrophoretic medium to an electric field resulting in the electrophoretic migration of the test sample through the medium, under conditions and time sufficient for the target molecules in the test sample to specifically bind to the capture probes, thereby forming target molecule/capture probe complexes, and for non-target molecules to migrate through and elute from the medium, wherein only target molecules bind to the capture probes and are immobilized in the medium;
- d) treating the electrophoretic medium to release at least one of the following:
- i) the one or more capture probe,
- ii) the target molecules, or
- iii) the target/capture probe binding-complexes;
- and
- e) eluting the target molecules or target/capture probe complexes from the medium, whereby the released and eluted target molecules are substantially pure.
6 more elements
A method of detecting one or more target molecules in a test sample, comprising the steps of:
- a) copolymerizing one or more aptamer capture probes with a material capable of forming a matrix suitable for electrophoresis, wherein said copolymerization forms an electrophoretic medium containing immobilized aptamer capture probes covalently attached to the electrophoretic medium, and wherein said one or more aptamer capture probes are modified with a monomer capable of forming a matrix suitable for electrophoresis, and wherein the aptamer capture probe is a thrombin-binding DNA aptamer;
- b) introducing a test sample into the electrophoretic medium;
- c) subjecting the electrophoretic medium to an electric field resulting in the electrophoretic migration of the test sample into the electrophoretic medium under conditions suitable for the target molecules to bind to the at least one immobilized aptamer capture probe, wherein the target molecule is human thrombin and is SEQ ID No.1;
- and
- d) detecting the presence or absence of target molecule/aptamer capture probe complexes immobilized in the medium,
- thereby detecting the presence or absence of one or more target molecules in the test sample.
3 more elements
A method of detecting one or more target molecules in a test sample, comprising the steps of:
- a) copolymerizing one or more aptamer capture probes with a material capable of forming a matrix suitable for electrophoresis, wherein said copolymerization forms an electrophoretic medium containing immobilized aptamer capture probes covalently attached to the electrophoretic medium, and wherein said one or more aptamer capture probes are modified with a monomer capable of forming a matrix suitable for electrophoresis;
- b) introducing a test sample into the electrophoretic medium;
- c) subjecting the electrophoretic medium to an electric field resulting in the electrophoretic migration of the test sample into the electrophoretic medium under conditions suitable for the target molecules to bind to the at least one immobilized aptamer capture probe, wherein the target molecule is selected from the group consisting of:
- SEQ ID No.2 and SEQ ID No.3;
- and
- d) detecting the presence or absence of target molecule/aptamer capture probe complexes immobilized in the medium,
- thereby detecting the presence or absence of one or more target molecules in the test sample.
4 more elements
A method for purifying molecules in a complex test sample comprising target molecules and non-target molecules, comprising:
- a) copolymerizing one or more capture probes with a material capable of forming a matrix suitable for electrophoresis, wherein said copolymerization forms an electrophoretic medium containing immobilized capture probes covalently attached to the electrophoretic medium, and wherein said one or more capture probes are modified with a monomer capable of forming a matrix suitable for electrophoresis, and wherein said capture probes are selected from the group consisting of a nucleic acid, modified nucleic acid and a nucleic acid analog, and wherein one or more capture probes are immobilized in a two dimensional array within the electrophoretic medium;
- b) introducing a test sample into the electrophoretic medium;
- c) subjecting the electrophoretic medium to an electric field resulting in the electrophoretic migration of the test sample through the medium, under conditions and time sufficient for the target molecules to specifically bind to the capture probes, thereby forming target molecule/capture probe complexes, and allowing non-target molecules to migrate through and elute from the medium, wherein only target molecules bind to the capture probes and are immobilized in the medium,
- thereby subtracting out the target molecule from the sample preparation and purifying molecules in the complex test sample.
1 more element
A method of detecting the presence or absence of one or more target molecules in a test sample, wherein the one or more target molecules has a defined mobility in an appropriate electrophoretic medium when the medium is exposed to an electric field and the target molecule can form binding complexes with capture probes, comprising the steps of:
- a) copolymerizing one or more capture probes with a material capable of forming a matrix suitable for electrophoresis to form an electrophoretic medium containing immobilized capture probes covalently attached to the electrophoretic medium, wherein said one or more capture probes are modified with a 5′-acrylamide moiety and are selected from the group consisting of a nucleic acid, modified nucleic acid and a nucleic acid analog, and wherein said one or more capture probes are immobilized in a three dimensional array within the electrophoretic medium;
- b) introducing a test sample into the electrophoretic medium;
- c) subjecting the electrophoretic medium to an electric field resulting in the electrophoretic migration of the test sample into the region or regions of the electrophoretic medium containing immobilized capture probes, under conditions suitable for the target molecules to bind to the immobilized probes;
- and
- d) detecting the presence, or absence of target molecules or target molecule/capture probe complexes immobilized in the medium,
- thereby detecting the presence or absence of one or more target molecules in the test sample.
3 more elements
A method for purifying or concentrating target molecules from a complex test sample, comprising:
- a) copolymerizing one or more capture probes modified with a 5′-acrylamide moiety and selected from the group consisting of a nucleic acid, modified nucleic acid and a nucleic acid analog with a material capable of forming a matrix suitable for electrophoresis to form an electrophoretic medium containing immobilized capture probes covalently attached to the electrophoretic medium, wherein said one or more capture probes are immobilized in a three dimensional array within the electrophoretic medium;
- b) introducing a test sample into the electrophoretic medium;
- c) subjecting the electrophoretic medium to an electric field resulting in the electrophoretic migration of the test sample through the medium, under conditions and time sufficient for the target molecules in the test sample to specifically bind to the capture probes, thereby forming target molecule/capture probe complexes, and for non-target molecules to migrate through and elute from the medium, wherein only target molecules bind to the capture probes and are immobilized in the medium;
- d) treating the electrophoretic medium to release at least one of the following:
- i) the one or more capture probes,
- ii) the target molecules, or
- iii) the target/capture probe binding complexes;
- and
- e) eluting the target molecules or target/capture probe complexes from the medium, whereby the released and eluted target molecules are substantially pure.
6 more elements
A method for purifying molecules in a complex test sample comprising target molecules and non-target molecules, comprising:
- a) copolymerizing one or more capture probes modified with a 5′-acrylamide moiety and selected from the group consisting of a nucleic acid, modified nucleic acid and a nucleic acid analog with a material capable of forming a matrix suitable for electrophoresis to form an electrophoretic medium containing immobilized capture probes covalently attached to the electrophoretic medium, wherein said one or more capture probes are immobilized in a three dimensional array within the electrophoretic medium;
- b) introducing a test sample into the electrophoretic medium;
- c) subjecting the electrophoretic medium to an electric field resulting in the electrophoretic migration of the test sample through the medium, under conditions and time sufficient for the target molecules in the test sample to specifically bind to the capture probes, thereby forming target molecule/capture probe complexes and allowing non-target molecules to migrate through and elute from the medium, wherein only target molecules bind to the capture probes and are immobilized in the medium,
- thereby subtracting out the target molecule from the sample preparation and purifying molecules in the complex test sample.
1 more element
A method of detecting the presence or absence of one or more target molecules in a test sample, wherein the one or more target molecules have a defined mobility in an appropriate electrophoretic medium when the medium is exposed to an electric field and the one or more target molecules can form binding complexes with capture probes, comprising the steps of:
- a) copolymerizing one or more capture probes with a material capable of forming a matrix suitable for electrophoresis, wherein said copolymerization forms an electrophoretic medium containing immobilized capture probes covalently attached to the electrophoretic medium, and wherein said one or more capture probes are modified with a monomer capable of forming a matrix suitable for electrophoresis, and wherein said capture probes are selected from the group consisting of a nucleic acid, modified nucleic acid and a nucleic acid analog, and wherein one or more capture probes are immobilized in a three dimensional array within the electrophoretic medium,
- b) introducing a test sample into the electrophoretic medium;
- c) subjecting the electrophoretic medium to an electric field resulting in the electrophoretic migration of the test sample into the region or regions of the electrophoretic medium containing immobilized capture probes, under conditions suitable for the target molecules to bind to the immobilized probes;
- and
- d) detecting the presence or absence of target molecules or target molecule/capture probe complexes immobilized in the medium,
- thereby detecting the presence or absence of one or more target molecules in the test sample.
3 more elements
A method for purifying or concentrating target molecules from a complex test sample, comprising:
- a) copolymerizing one or more capture probes with a material capable of forming a matrix suitable for electrophoresis, wherein said copolymerization forms an electrophoretic medium containing immobilized capture probes covalently attached to the electrophoretic medium, and wherein said one or more capture probes are modified with a monomer capable of forming a matrix suitable for electrophoresis, and wherein said capture probes are selected from the group consisting of a nucleic acid, modified nucleic acid and a nucleic acid analog, and wherein one or more capture probes are immobilized in a three dimensional array within the electrophoretic medium,
- b) introducing a test sample into the electrophoretic medium;
- c) subjecting the electrophoretic medium to an electric field resulting in the electrophoretic migration of the test sample through the medium, under conditions and time sufficient for the target molecules in the test sample to specifically bind to the capture probes, thereby forming target molecule/capture probe complexes, and for non-target molecules to migrate through and elute from the medium, wherein only target molecules bind to the capture probes and are immobilized in the medium;
- d) treating the electrophoretic medium to release at least one of the following:
- i) the one or more capture probe,
- ii) the target molecules, or
- iii) the target/capture probe binding-complexes;
- and
- e) eluting the target molecules or target/capture probe complexes from the medium, whereby the released and eluted target molecules are substantially pure.
6 more elements
A method for purifying molecules in a complex test sample comprising target molecules and non-target molecules, comprising:
- a) copolymerizing one or more capture probes with a material capable of forming a matrix suitable for electrophoresis, wherein said copolymerization forms an electrophoretic medium containing immobilized capture probes covalently attached to the electrophoretic medium, and wherein said one or more capture probes are modified with a monomer capable of forming a matrix suitable for electrophoresis, and wherein said capture probes are selected from the group consisting of a nucleic acid, modified nucleic acid and a nucleic acid analog, and wherein one or more capture probes are immobilized in a three dimensional array within the electrophoretic medium;
- b) introducing a test sample into the electrophoretic medium;
- c) subjecting the electrophoretic medium to an electric field resulting in the electrophoretic migration of the test sample through the medium, under conditions and time sufficient for the target molecules to specifically bind to the capture probes, thereby forming target molecule/capture probe complexes, and allowing non-target molecules to migrate through and elute from the medium, wherein only target molecules bind to the capture probes and are immobilized in the medium,
- thereby subtracting out the target molecule from the sample preparation and purifying molecules in the complex test sample.
1 more element
Dependencies are inferred from the claim text.