Nucleic acid detection method involving the direct generation of a measurable signal
First Claim
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1. A method for detecting a target nucleic acid in a sample, comprising the following steps:
- (1) contacting a sample with a first oligonucleotide primer which hybridizes with said target nucleic acid to leave one or more non-complementary nucleotides unpaired at the 3′
end of said first oligonucleotide primer, wherein said first oligonucleotide primer contains a fluorophore quencher at its 5′
-end and a fluorophore at its 3′
-end, or a fluorophore quencher at its 3′
-end and a fluorophore at its 5′
-end, and wherein the 3′
-nucleotide of said first oligonucleotide primer contains a free hydroxyl group;
(2) contacting said sample with at least one additional primer, a mixture of nucleotides, and a polymerase enzyme having 3′
-5′
nuclease activity;
(3) subjecting the sample containing said first oligonucleotide primer, said mixture of nucleotides, said at least one additional primer, and said enzyme, to real-time PCR; and
(4) determining whether a measurable signal is produced by said fluorophore during said real-time PCR;
wherein when said target nucleic acid is present in said sample, said first oligonucleotide primer hybridizes with the target nucleic acid so that nucleic acid extension occurs simultaneously with cleavage of one or more of said non-complementary nucleotides by said enzyme, producing a measurable signal by said fluorophore.
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Abstract
A nucleic acid detection method involving the direct generation of a measurable signal, by the action of an enzyme with 3′-5′ nuclease activity, and the applications for it, the signal generated can be detectable and quantifiable and can be carried out in real-time, in this method, the nucleic acid is placed in contact with at least one oligonucleotide that does not hybridize perfectly with it, so that the enzyme will split it at the unpaired bases generating the signal, the oligonucleotide can be labeled.
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Citations
23 Claims
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1. A method for detecting a target nucleic acid in a sample, comprising the following steps:
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(1) contacting a sample with a first oligonucleotide primer which hybridizes with said target nucleic acid to leave one or more non-complementary nucleotides unpaired at the 3′
end of said first oligonucleotide primer, wherein said first oligonucleotide primer contains a fluorophore quencher at its 5′
-end and a fluorophore at its 3′
-end, or a fluorophore quencher at its 3′
-end and a fluorophore at its 5′
-end, and wherein the 3′
-nucleotide of said first oligonucleotide primer contains a free hydroxyl group;(2) contacting said sample with at least one additional primer, a mixture of nucleotides, and a polymerase enzyme having 3′
-5′
nuclease activity;(3) subjecting the sample containing said first oligonucleotide primer, said mixture of nucleotides, said at least one additional primer, and said enzyme, to real-time PCR; and (4) determining whether a measurable signal is produced by said fluorophore during said real-time PCR; wherein when said target nucleic acid is present in said sample, said first oligonucleotide primer hybridizes with the target nucleic acid so that nucleic acid extension occurs simultaneously with cleavage of one or more of said non-complementary nucleotides by said enzyme, producing a measurable signal by said fluorophore. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 22)
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10. A method for detecting a target nucleic acid in a sample, comprising the following steps:
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(1) contacting a sample with an oligonucleotide which hybridizes with said target nucleic acid to leave one or more non-complementary nucleotides unpaired at the 3′
end of said oligonucleotide, wherein said oligonucleotide contains a fluorophore quencher at the 5′
-end of said oligonucleotide and a fluorophore at the 3′
-end of said oligonucleotide, or a fluorophore quencher at the 3′
-end of said oligonucleotide and a fluorophore at the 5′
-end of said oligonucleotide, and wherein the 3′
-nucleotide of said oligonucleotide contains a free hydroxyl group;(2) contacting said sample with an enzyme having 3′
-5′
nuclease activity, a mixture of nucleotides, at least two primers, an enzyme with polymerase activity, and an enzyme with chain displacement activity, 5′
-3′
nuclease activity, or both; and(3) determining whether a measurable signal is produced by said fluorophore; wherein when said target nucleic acid is present in said sample, said oligonucleotide hybridizes with the target nucleic acid and functions as a probe to generate a measurable signal without acting as a primer for an extension reaction. - View Dependent Claims (15)
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13. A method for detecting a target nucleic acid in a sample, comprising the following steps:
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(1) contacting a sample with an oligonucleotide which hybridizes with said target nucleic acid to leave one or more non-complementary nucleotides unpaired at the 3′
end of said oligonucleotide, wherein said oligonucleotide contains a fluorophore quencher at the 5′
-end of said oligonucleotide and a fluorophore at the 3′
-end of said oligonucleotide, or a fluorophore quencher at the 3′
-end of said oligonucleotide and a fluorophore at the 5′
-end of said oligonucleotide, wherein the 3′
-nucleotide of said oligonucleotide contains a free hydroxyl group, and wherein said oligonucleotide contains modifications in the binding of its bases by non-phosphodiester bonds or by the inclusion of spacers, to protect these positions from 3′
-5′
nuclease activity;(2) contacting said sample with an enzyme having 3′
-5′
nuclease activity; and(3) determining whether a measurable signal is produced by said fluorophore; wherein when said target nucleic acid is present in said sample, said one or more non-complementary nucleotides unpaired at the 3′
end of said oligonucleotide are cleaved by said enzyme, producing a measurable signal by said fluorophore. - View Dependent Claims (14)
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16. A method for detecting a target nucleic acid in a sample, comprising the following steps:
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(1) contacting a sample with an oligonucleotide which hybridizes with said target nucleic acid to leave one or more non-complementary nucleotides unpaired at the 3′
end of said oligonucleotide, wherein said oligonucleotide contains a fluorophore quencher at the 5′
-end of said oligonucleotide and a fluorophore at the 3′
-end of said oligonucleotide, or a fluorophore quencher at the 3′
-end of said oligonucleotide and a fluorophore at the 5′
-end of said oligonucleotide, and wherein the 3′
-nucleotide of said oligonucleotide contains a free hydroxyl group;(2) contacting said sample with an enzyme having 3′
-5′
nuclease activity; and(3) determining whether a measurable signal is produced by said fluorophore; wherein said method is performed in a hybridization system in the absence of a polymerization reaction, and wherein when said target nucleic acid is present in said sample, said one or more non-complementary nucleotides unpaired at the 3′
end of said oligonucleotide are cleaved by said enzyme, producing a measurable signal by said fluorophore. - View Dependent Claims (17)
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18. A kit for determining a nucleic acid sequence in a sample, wherein said kit comprises an oligonucleotide that contains a sequence complementary to the nucleic acid sequence to be determined, but with one or more non-complementary bases at the 3′
- end of the oligonucleotide, wherein said oligonucleotide contains a fluorophore quencher at the 5′
-end of said oligonucleotide and a fluorophore at the 3′
-end of said oligonucleotide, or a fluorophore quencher at the 3′
-end of said oligonucleotide and a fluorophore at the 5′
-end of said oligonucleotide, wherein the 3′
-nucleotide of said oligonucleotide contains a free hydroxyl group, and wherein said oligonucleotide is modified at its 3′
end to protect the hydrolysis of certain positions, and wherein said kit further comprises a polymerase enzyme having 3′
-5′
nuclease proofreading activity. - View Dependent Claims (19, 20, 21, 23)
- end of the oligonucleotide, wherein said oligonucleotide contains a fluorophore quencher at the 5′
Specification