Detection of small RNAS
First Claim
1. A method for detecting a small RNA, the method comprising:
- (1) forming a mixture comprising (a) a sample suspected of comprising the small RNA;
(b) a ligating agent; and
(c) a target probe set for detecting the small RNA, the target probe set comprising (i) a first target probe comprising a 3′
portion that hybridizes to the small RNA and a 5′
portion having a first PCR primer target sequence, and (ii) a second target probe comprising a 5′
portion that hybridizes to the RNA immediately adjacent to the 3′
end of the first target probe and a 3′
portion having a second PCR primer target sequence, wherein the mixture is formed under conditions in which a target probe set hybridizes to the RNA and ligates to form a probe set ligation sequence;
(2) forming a detection mixture comprising a probe set ligation sequence, a first PCR primer which hybridizes to the complement of the first PCR primer target, and a second PCR primer which hybridizes to the second PCR primer target;
(3) amplifying any probe set ligation sequence comprised by the detection mixture using a polymerase chain reaction; and
(4) detecting amplification of any probe set ligation sequence comprised by the detection mixture, wherein the small RNA comprises a sequence consisting of from about 20 to about 40 contiguous nucleotides.
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Accused Products
Abstract
Methods for detecting an RNA such as a small RNA comprising up to about 40 nucleotides are disclosed. The methods comprise forming a ligation mixture comprising a sample suspected of comprising the small RNA, a ligase and a target probe set. The target probe set comprises a first target probe comprising a 3′ portion that hybridizes to the small RNA and a 5′ portion having a first PCR primer target sequence, and a second target probe comprising a 5′ portion that hybridizes to the RNA immediately adjacent to the 3′ end of the first target probe and a 3′ portion having a second PCR primer target sequence. The target probe set hybridizes to the RNA and ligates to form a probe set ligation sequence. A probe set ligation sequence can be amplified and detected using a polymerase chain reaction.
62 Citations
60 Claims
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1. A method for detecting a small RNA, the method comprising:
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(1) forming a mixture comprising (a) a sample suspected of comprising the small RNA;
(b) a ligating agent; and
(c) a target probe set for detecting the small RNA, the target probe set comprising (i) a first target probe comprising a 3′
portion that hybridizes to the small RNA and a 5′
portion having a first PCR primer target sequence, and (ii) a second target probe comprising a 5′
portion that hybridizes to the RNA immediately adjacent to the 3′
end of the first target probe and a 3′
portion having a second PCR primer target sequence, wherein the mixture is formed under conditions in which a target probe set hybridizes to the RNA and ligates to form a probe set ligation sequence;
(2) forming a detection mixture comprising a probe set ligation sequence, a first PCR primer which hybridizes to the complement of the first PCR primer target, and a second PCR primer which hybridizes to the second PCR primer target;
(3) amplifying any probe set ligation sequence comprised by the detection mixture using a polymerase chain reaction; and
(4) detecting amplification of any probe set ligation sequence comprised by the detection mixture, wherein the small RNA comprises a sequence consisting of from about 20 to about 40 contiguous nucleotides. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18)
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19. A method for detecting a small RNA in a sample, the method comprising:
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(1) forming a mixture comprising (a) a sample suspected of comprising the small RNA;
(b) a target probe set for detecting the small RNA, the target probe set comprising (i) a first target probe comprising a 3′
portion that hybridizes to the small RNA and a 5′
portion having a first PCR primer target sequence, and (ii) a second target probe comprising a 5′
portion that hybridizes to the RNA immediately adjacent to the 3′
end of the first target probe and a 3′
portion having a second PCR primer target sequence, wherein one of the first or second target probes further comprises an affinity tag; and
(c) an affinity tag binding partner which is covalently attached to a solid phase support, wherein upon forming the mixture, a complex forms comprising the small RNA hybridized to the first target probe and the second target probe, and the affinity tag binding partner bound to the affinity tag;
(2) removing unhybridized first target probe and unhybridized second target probe from the mixture;
(3) forming a probe set ligation sequence by contacting the complex with a ligating agent;
(4) forming a detection mixture comprising the probe set ligation sequence, a first PCR primer which hybridizes to the complement of the first PCR primer target, and a second PCR primer which hybridizes to the second PCR primer target;
(5) amplifying any probe set ligation sequence; and
(6) detecting amplification of any probe set ligation sequence comprised by the mixture. - View Dependent Claims (20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39)
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40. A small RNA detection kit comprising:
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a first target probe comprising a 3′
portion that hybridizes to a small RNA and a 5′
portion having a first PCR primer target sequence;
a second target probe comprising a 5′
portion that hybridizes to the small RNA immediately adjacent to the 3′
portion of the first target probe and a 3′
portion having a second PCR primer target sequence, wherein upon hybridization of the first and second target probes to the small RNA in the presence of a ligase, a ligation sequence is formed, packaged in a container. - View Dependent Claims (41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60)
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Specification