Nucleic acid detection using structured probes
First Claim
1. A method of detecting target nucleic acid sequences, the method comprising(a) bringing into contact one or more target samples and one or more structured probes, and incubating under conditions that promote hybridization between target nucleic acid sequences in the target samples and the structured probes, wherein each structured probe is bifunctional, wherein one function is as a probe to a target nucleic acid sequence and the other function is a detection function, wherein the detection function is not possible unless the probe hybridizes to the target nucleic acid sequence, wherein each structured probe has a first end and a second end, wherein each structured probe comprises at least two complementary portions, a target probe portion, and a detection portion, wherein the two or more complementary portions comprise a first complementary portion and a second complementary portion, wherein two or more of the complementary portions form a duplex region, wherein formation of the duplex region forms a loop, wherein at least a portion of the target probe portion is in the loop, wherein hybridization of the target probe portion to the target nucleic acid sequence disrupts the duplex region, (b) detecting the structured probes, wherein, prior to detection, the structured probes are treated to alter unhybridized probes, wherein only probes with intact duplex regions are altered by treatment of the structured probes, wherein altered probes are not detected.
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Abstract
Disclosed are compositions and a method for detection of nucleic acid sequences. The disclosed method uses a structured probe to distinguish between sequences. Structured probes are bifunctional molecules where one function is as a probe to a target nucleic acid sequence and the other function is as a detection sequence to facilitate detection of the probe. Structured probes include a detection sequence, sequence complementary to a target sequence, and sequences that form duplex regions (higher order structures). The duplex region is stable unless the probe hybridizes to the target sequence. The disclosed method involves hybridizing the structured probe to a target sequence and detecting the detection sequence on the structured probe. The detection sequence is available for detection only if the duplex region of the structured probe is disrupted. This links detection of the structured probe with the hybridization of the structured probe to the target sequence.
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Citations
56 Claims
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1. A method of detecting target nucleic acid sequences, the method comprising
(a) bringing into contact one or more target samples and one or more structured probes, and incubating under conditions that promote hybridization between target nucleic acid sequences in the target samples and the structured probes, wherein each structured probe is bifunctional, wherein one function is as a probe to a target nucleic acid sequence and the other function is a detection function, wherein the detection function is not possible unless the probe hybridizes to the target nucleic acid sequence, wherein each structured probe has a first end and a second end, wherein each structured probe comprises at least two complementary portions, a target probe portion, and a detection portion, wherein the two or more complementary portions comprise a first complementary portion and a second complementary portion, wherein two or more of the complementary portions form a duplex region, wherein formation of the duplex region forms a loop, wherein at least a portion of the target probe portion is in the loop, wherein hybridization of the target probe portion to the target nucleic acid sequence disrupts the duplex region, (b) detecting the structured probes, wherein, prior to detection, the structured probes are treated to alter unhybridized probes, wherein only probes with intact duplex regions are altered by treatment of the structured probes, wherein altered probes are not detected.
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55. A method of detecting target nucleic acid sequences, the method comprising
(a) bringing into contact one or more target samples and one or more structured probes, and incubating under conditions that promote hybridization between target nucleic acid sequences in the target samples and the structured probes, wherein each structured probe is bifunctional, wherein one function is as a probe to a target nucleic acid sequence and the other function is a detection function, wherein the detection function is not possible unless the probe hybridizes to the target nucleic acid sequence, wherein each structured probe has a first end and a second end, wherein each structured probe comprises at least two complementary portions, a target probe portion, and a detection portion, wherein the two or more complementary portions comprise a first complementary portion and a second complementary portion, wherein two or more of the complementary portions form a duplex region, wherein formation of the duplex region forms a loop, wherein at least a portion of the target probe portion is in the loop, wherein hybridization of the target probe portion to the target nucleic acid sequence disrupts the duplex region, (b) detecting the structured probes, wherein, prior to detection, the structured probes are treated to alter unhybridized probes, wherein the treatment is extending the second end of the probe using probe sequence as template, wherein only probes with intact duplex regions are altered by treatment of the structured probes, wherein altered probes are not detected.
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56. A method of detecting target nucleic acid sequences, the method comprising
(a) bringing into contact one or more target samples and one or more structured probes, and incubating under conditions that promote hybridization between target nucleic acid sequences in the target samples and the structured probes, wherein each structured probe is bifunctional, wherein one function is as a probe to a target nucleic acid sequence and the other function is a detection function, wherein the detection function is not possible unless the probe hybridizes to the target nucleic acid sequence, wherein each structured probe has a first end and a second end, wherein each structured probe comprises at least two complementary portions, a target probe portion, and a detection portion, wherein the two or more complementary portions comprise a first complementary portion and a second complementary portion, wherein two or more of the complementary portions interact with each other to form a duplex region, wherein formation of the duplex region forms a loop, wherein at least a portion of the target probe portion is in the loop, wherein hybridization of the target probe portion to the target nucleic acid sequence disrupts the duplex region, (b) detecting the structured probes, wherein, prior to detection, the structured probes are treated to alter unhybridized probes, wherein only probes with the duplex region intact are altered by the treatment of the structured probes, whereby altered probes are not detected.
Specification