Direct SNP detection with unamplified DNA
First Claim
1. A method for detecting a target nucleic acid sequence in a sample, the sample comprising nucleic acid molecules of higher biological complexity relative to amplified nucleic acid molecules and the target nucleic acid sequence differs from one or more nucleic acid sequences by at least one nucleotide, the method comprising the steps of:
- a) providing an addressable substrate having a capture oligonucleotide bound thereto, wherein the capture oligonucleotide has a sequence that is complementary to at least part of a first portion of the target nucleic acid sequence;
b) providing a detection probe comprising detector oligonucleotides, wherein the detector oligonucleotides have a sequence that is complementary to at least part of a second portion of the target nucleic acid sequence of step (a);
c) contacting the sample with the substrate and the detection probe under conditions that are effective for the hybridization of the capture oligonucleotide to the first portion of the target nucleic acid sequence and the hybridization of the detection probe to the second portion of the target nucleic acid sequence and to allow for discrimination between the target and said one or more nucleic acid sequences that differ by at least one nucleotide; and
d) detecting whether the capture oligonucleotide and detection probe hybridized with the first and second portions of the target nucleic acid sequence.
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Abstract
The present invention provides methods for detecting a target nucleic acid molecule in a sample that comprises nucleic acid molecules of higher biological complexity than that of amplified nucleic acid molecules. In particular, the present invention provides methods and probes for detecting a single nucleotide polymorphism (SNP) in a sample that comprises nucleic acid molecules of higher biological complexity than that of amplified nucleic acid molecules.
85 Citations
167 Claims
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1. A method for detecting a target nucleic acid sequence in a sample, the sample comprising nucleic acid molecules of higher biological complexity relative to amplified nucleic acid molecules and the target nucleic acid sequence differs from one or more nucleic acid sequences by at least one nucleotide, the method comprising the steps of:
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a) providing an addressable substrate having a capture oligonucleotide bound thereto, wherein the capture oligonucleotide has a sequence that is complementary to at least part of a first portion of the target nucleic acid sequence;
b) providing a detection probe comprising detector oligonucleotides, wherein the detector oligonucleotides have a sequence that is complementary to at least part of a second portion of the target nucleic acid sequence of step (a);
c) contacting the sample with the substrate and the detection probe under conditions that are effective for the hybridization of the capture oligonucleotide to the first portion of the target nucleic acid sequence and the hybridization of the detection probe to the second portion of the target nucleic acid sequence and to allow for discrimination between the target and said one or more nucleic acid sequences that differ by at least one nucleotide; and
d) detecting whether the capture oligonucleotide and detection probe hybridized with the first and second portions of the target nucleic acid sequence. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161)
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38. A method for detecting one or more target nucleic acid sequences in a sample, the sample comprising nucleic acid molecules of higher biological complexity relative to amplified nucleic acid molecules and the one or more target nucleic acid sequences each differ from known nucleic acid sequences by at least one nucleotide, the method comprising the steps of:
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a) providing an addressable substrate having a plurality of capture oligonucleotides bound thereto, wherein the capture oligonucleotides have sequences that are complementary to one or more portions of the one or more target nucleic acid sequences;
b) providing one or more detector probes comprising detector oligonucleotides, wherein the detector oligonucleotides have sequences that are complementary to one or more portions of the one or more target nucleic acid sequences of step (a) that are not recognized by a capture oligonucleotide on the substrate;
c) contacting the sample with the substrate and the detector probes under conditions that are effective for the hybridization of the capture oligonucleotides to one or more portions of the one or more target nucleic acid sequences and the hybridization of the detector probes to portions of the one or more target nucleic acid sequences that are not recognized by a capture oligonucleotide and to allow for discrimination between targets that differ by at least one nucleotide; and
d) detecting whether any of the capture oligonucleotide and detector probes hybridized with any of the target nucleic acid sequences. - View Dependent Claims (39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 162, 163, 164, 165, 166, 167)
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75. A method for identifying a single nucleotide polymorphism in a sample, the sample comprising nucleic acid molecules of higher biological complexity relative to amplified nucleic acid molecules, the method comprising the steps of:
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a) providing an addressable substrate having at least one capture oligonucleotide bound thereto, wherein the at least one capture oligonucleotide has a sequence that is complementary to at least a part of a nucleic acid target that comprises a specific polymorphism;
b) providing a detector probe having detector oligonucleotides bound thereto, wherein the detector oligonucleotides has a sequence that is complementary to at least a portion of the nucleic acid target of step (a);
c) contacting the sample with the substrate and the detector probe under conditions that are effective for the hybridization of the capture oligonucleotide to the nucleic acid target and the hybridization of the detector probe to the nucleic acid target and to allow for discrimination between targets that differ by a single nucleotide; and
d) detecting whether the capture oligonucleotide and detector probe hybridized with the nucleic acid target. - View Dependent Claims (76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110)
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111. A method for identifying one or more single nucleotide polymorphisms in a sample, the sample comprising nucleic acid molecules of higher biological complexity relative to amplified nucleic acid molecules, the method comprising the steps of:
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a) providing an addressable substrate having a plurality of capture oligonucleotides bound thereto, wherein the capture oligonucleotides have sequences that are complementary to multiple portions of a nucleic acid target, each said portion comprising a specific polymorphism;
b) providing one or more detector probes comprising detector oligonucleotides, wherein the detector oligonucleotides have a sequence that is complementary to at least a portion of one of the nucleic acid targets of step (a) that is not recognized by a capture oligonucleotide on the substrate;
c) contacting the sample with the substrate and the detector probes under conditions that are effective for the hybridization of the capture oligonucleotides to multiple portions of the nucleic acid target and the hybridization of the detector probe to the nucleic acid target and to allow for discrimination between targets that differ by a single nucleotide; and
d) detecting whether any of the capture oligonucleotides and detector probes hybridized with any of the nucleic acid targets. - View Dependent Claims (112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145)
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146. The method of claim 146, wherein the substrate is contacted with silver stain to produce the change in conductivity.
Specification