[미국특허]
Molecular beacon-based methods for detection of targets using abscription
원문보기
IPC분류정보
국가/구분
United States(US) Patent
등록
국제특허분류(IPC7판)
C12Q-001/70
C12P-019/34
출원번호
US-0502217
(2009-07-13)
등록번호
US-8211644
(2012-07-03)
발명자
/ 주소
Hanna, Michelle M.
McCarthy, David
출원인 / 주소
RiboMed Biotechnologies, Inc.
대리인 / 주소
The Law Office of Jane K. Babin, Professional Corporation
인용정보
피인용 횟수 :
4인용 특허 :
43
초록▼
The present invention provides methods for detecting targets using an Abscription assay that exploits molecular beacon-based detection technology. The methods of the invention are highly sensitive and can be performed on a NanoDrop scale and can be multiplexed for simultaneous detection of multiple
The present invention provides methods for detecting targets using an Abscription assay that exploits molecular beacon-based detection technology. The methods of the invention are highly sensitive and can be performed on a NanoDrop scale and can be multiplexed for simultaneous detection of multiple targets.
대표청구항▼
1. A method for detecting a target comprising: a) performing an Abscription reaction synthesizing at least one Abscript, wherein synthesizing the at least one Abscript indicates the presence of a target;b) hybridizing the at least one Abscript synthesized in step a) to at least one molecular beacon,
1. A method for detecting a target comprising: a) performing an Abscription reaction synthesizing at least one Abscript, wherein synthesizing the at least one Abscript indicates the presence of a target;b) hybridizing the at least one Abscript synthesized in step a) to at least one molecular beacon, wherein the molecular beacon comprises a probe sequence complementary to the Abscript, thereby forming an Abscript:beacon hybrid;c) contacting the Abscript:beacon hybrid with a polymerase and nucleotides, thereby extending the Abscript in the Abscript:beacon hybrid and opening the molecular beacon; andd) detecting fluorescence from the opened molecular beacon, thereby detecting the target. 2. The method of claim 1, wherein the target is a protein or a nucleic acid. 3. The method of claim 1, wherein the at least one Abscript is an RNA sequence of 6-20 nucleotides in length. 4. The method of claim 3, wherein the at least one Abscript is 11 nucleotides in length. 5. The method of claim 1, wherein the steps a), b) and c) are performed in a single reaction. 6. A method for detecting a target comprising: a) performing an Abscription reaction synthesizing an Abscript, wherein synthesizing the Abscript indicates the presence of a target;b) hybridizing the Abscript synthesized in step a) to at least on first molecule of a molecular beacon, wherein the molecular beacon comprises a probe sequence complementary to the Abscript, thereby forming an Abscript:beacon hybrid;c) contacting the Abscript:beacon hybrid with a DNA polymerase and deoxyribonucleotides, thereby extending the Abscript in the Abscript: beacon hybrid, wherein extending the Abscript produces a double-strand DNA sequence recognized by a restriction enzyme;d) digesting the double-strand DNA sequence with the restriction enzyme, thereby producing a fluorophore fragment and an extended Abscript fragment; ande) detecting fluorescence from the fluorophore fragment, thereby detecting the target. 7. The method of claim 6, wherein the restriction enzyme is selected from HaeII, HhaI, HinPI and Hpy188I. 8. The method of claim 3, further comprising: f) hybridizing the extended Abscript fragment of step d) with a second molecule of the molecular beacon, thereby forming an extended Abscript fragment:beacon hybrid;g) contacting the extended Abscript fragment:beacon hybrid with a DNA polymerase and deoxyribonucleotides, thereby re-extending the extended Abscript fragment in the extended Abscript fragment:probe hybrid, wherein extending the extended Abscript fragment produces a double-strand DNA sequence recognized by the restriction enzyme;h) digesting the double-strand DNA sequence with the restriction enzyme, thereby producing a fluorophore fragment and an extended Abscript fragment;i) repeating steps f) through h); andj) detecting fluorescence of the fluorophore fragment, thereby detecting the target. 9. The method of claim 8, wherein the steps a) through i) are performed in a single reaction. 10. The method of claim 8, wherein steps f) through h) are repeated at least 10 times. 11. A method for detecting at least two targets comprising: a) performing a first Abscription reaction synthesizing a first Abscript, wherein synthesizing the first Abscript indicates the presence of a first target;b) performing a second Abscription reaction synthesizing a second Abscript having a different nucleotide sequence than the first Abscript, wherein synthesizing the second Abscript indicates the presence of a second target;c) hybridizing the first Abscript synthesized in step a) with a first molecular beacon comprising a first fluorophore, wherein the first molecular beacon comprises a probe sequence complementary to the first Abscript, thereby forming a first Abscript:beacon hybrid;d) -hybridizing the second Abscript synthesized in step b) with a second molecular beacon comprising a second fluorphore that fluoresces at a different wavelength from the first fluorophore, wherein the second molecular beacon comprises a probe sequence complementary to the second Abscript, thereby forming a second Abscript:beacon hybrid;e) contacting the first and second Abscript:beacon hybrids with a polymerase and nucleotides, thereby extending the first and second Abscripts in the Abscript:beacon hybrids and opening the first and second molecular beacons; andf) detecting fluorescence of the first and second opened molecular beacons, thereby detecting the two targets. 12. The method of claim 11, wherein steps a)-f) are performed in a single reaction. 13. A method of detecting a target in plurality of samples, comprising: a) performing an Abscription reaction for each of a plurality of samples, synthesizing at least one Abscript, wherein synthesizing the at least one Abscript indicates the presence of a target;b) hybridizing the at least one Abscript synthesized in step a) with at least one molecular beacon, wherein the molecular beacon comprises a probe sequence complementary to the Abscript to form an Abscript:beacon hybrid;c) contacting the Abscript:beacon hybrid with a polymerase and nucleotides, thereby extending the Abscripts in the Abscript:beacon hybrids thereby opening the molecular beacon; andd) detecting fluorescence of the opened molecular beacon, thereby detecting the target. 14. The method of claim 13, wherein each sample is from a different source and the same target is detected in each sample. 15. The method of claim 13, wherein each sample is the same, and the target detected in each sample is different. 16. The method of claim 1, wherein the polymerase is a DNA polymerase. 17. The method of claim 11, wherein the polymerase is a DNA polymerase. 18. The method of claim 13, wherein the polymerase is a DNA polymerase.
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