Detection of small nucleic acids
First Claim
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1. A method for analyzing microRNA, comprising:
- a) contacting microRNA comprising a 3′
portion comprising a 3′
terminal end and a 5′
portion comprising a 5′
terminal end with a first hairpin probe and a second hairpin probe to form an RNA detection structure, whereini) said first hairpin probe comprises a 3′
region that is complementary said 3′
portion of said microRNA, and a 5′
region that is not complementary to said microRNA, wherein a first portion of said 5′
region is complementary to a second portion of said 5′
region, wherein said first portion and said second portion of said 5′
region hybridize to each other to form a first duplex when said hairpin probe is hybridized to said microRNA, and wherein said first duplex and said 3′
region of said probe are within one nucleotide of each other; and
ii) said second hairpin probe comprises a 5′
region that is complementary to said 5′
portion of said microRNA and a 3′
region that is not complementary to said microRNA, wherein a first portion of said 3′
region is complementary to a second portion of said 3′
region, wherein said first portion and said second portion of said 3′
region hybridize to each other to form a second duplex when said hairpin probe is hybridized to said microRNA, and wherein said second duplex and said 5′
region of said probe are within one nucleotide of each other;
wherein, in said RNA detection structure, said microRNA and said first and second hairpin probes form a dumbbell structure,b) detecting formation of said RNA detection structure, wherein formation of said RNA detection structure is indicative of the presence of said microRNA.
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Abstract
The present invention relates to compositions and methods for the detection and characterization of interfering RNAs such as micro RNAs (miRNAs) and small interfering RNAs (siRNAs) and other short nucleic acid molecules. More particularly, the present invention relates to methods for the detection and quantitation of interfering RNA expression. The present invention further provides for the detection of variants and types of miRNAs and siRNAs.
39 Citations
48 Claims
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1. A method for analyzing microRNA, comprising:
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a) contacting microRNA comprising a 3′
portion comprising a 3′
terminal end and a 5′
portion comprising a 5′
terminal end with a first hairpin probe and a second hairpin probe to form an RNA detection structure, whereini) said first hairpin probe comprises a 3′
region that is complementary said 3′
portion of said microRNA, and a 5′
region that is not complementary to said microRNA, wherein a first portion of said 5′
region is complementary to a second portion of said 5′
region, wherein said first portion and said second portion of said 5′
region hybridize to each other to form a first duplex when said hairpin probe is hybridized to said microRNA, and wherein said first duplex and said 3′
region of said probe are within one nucleotide of each other; andii) said second hairpin probe comprises a 5′
region that is complementary to said 5′
portion of said microRNA and a 3′
region that is not complementary to said microRNA, wherein a first portion of said 3′
region is complementary to a second portion of said 3′
region, wherein said first portion and said second portion of said 3′
region hybridize to each other to form a second duplex when said hairpin probe is hybridized to said microRNA, and wherein said second duplex and said 5′
region of said probe are within one nucleotide of each other;wherein, in said RNA detection structure, said microRNA and said first and second hairpin probes form a dumbbell structure, b) detecting formation of said RNA detection structure, wherein formation of said RNA detection structure is indicative of the presence of said microRNA. - 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)
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26. A method for analyzing microRNA in a sample, comprising:
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a) contacting microRNA comprising a 3′
portion comprising a 3′
terminal end and a 5′
portion comprising a 5′
terminal end with a first hairpin probe and a second hairpin probe to form an RNA detection structure whereini) said first hairpin probe comprises a 3′
region that is complementary said 3′
portion of said microRNA, and a 5′
region that is not complementary to said microRNA, wherein a first portion of said 5′
region is complementary to a second portion of said 5′
region, wherein said first portion and said second portion of said 5′
region hybridize to each other to form a first duplex when said hairpin probe is hybridized to said microRNA, and wherein said first duplex and said 3′
region of said probe are within one nucleotide of each other; andii) said second hairpin probe comprises a 5′
region that is complementary to said 5′
portion of said microRNA and a 3′
region that is not complementary to said microRNA, wherein a first portion of said 3′
region is complementary to a second portion of said 3′
region, wherein said first portion and said second portion of said 3′
region hybridize to each other to form a second duplex when said hairpin probe is hybridized to said microRNA, and wherein said second duplex and said 5′
region of said probe are within one nucleotide of each other;wherein, in said RNA detection structure, said microRNA and said first and second hairpin probes form a dumbbell structure, b) reacting said RNA detection structure with a nucleic acid modifying enzyme to form an modified RNA detection structure; c) detecting formation of said modified RNA detection structure, wherein formation of said modified RNA detection structure is indicative of the presence of said microRNA, and wherein said detecting formation of said modified RNA detection structure comprises use of an amplification reaction. - View Dependent Claims (27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48)
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Specification