Encoded Nanopore Sensor for Multiplex Nucleic Acids Detection
First Claim
1. A set of probe molecules comprising at least a first probe molecule and a second probe molecule, wherein at least two probe molecules comprise:
- a) a capture domain that comprises a sequence with complementarity to a target nucleic acid;
b) a terminal extension that is covalently linked to the 5′
end, the 3′
end, or both the 5′
end and the 3′
end of the capture domain; and
c) at least one polymer label attached to at least one of the terminal extension(s),wherein the nucleic acid capture domain of the first probe molecule comprises a sequence with complementarity to first target nucleic acid and the nucleic acid capture domain of the second probe molecule comprises a sequence with complementarity to a second target nucleic acid,and wherein the polymer label of the first probe molecule is different from the polymer label of the second probe molecule and provide for independent detection of the first and second target nucleic acids in a nanopore system.
1 Assignment
0 Petitions
Accused Products
Abstract
The present invention provides a new and improved multiplexed oligonucleotide detection method based on the nanopore technology with one or more probes containing a sequence with complementarity to the target oligonucleotide, a terminal extension at the probe'"'"'s 3′ terminus, 5′ terminus, or both termini and a label attached to the terminus. The improved probes and probe sets enable sensitive, selective, and direct multiplex detection, differentiation and quantification of distinct target oligonucleotides such as miRNAs. The inventive detection method may also be employed as a non-invasive and cost-effective diagnostic method based on miRNA levels in the patient'"'"'s tissue sample.
-
Citations
52 Claims
-
1. A set of probe molecules comprising at least a first probe molecule and a second probe molecule, wherein at least two probe molecules comprise:
-
a) a capture domain that comprises a sequence with complementarity to a target nucleic acid; b) a terminal extension that is covalently linked to the 5′
end, the 3′
end, or both the 5′
end and the 3′
end of the capture domain; andc) at least one polymer label attached to at least one of the terminal extension(s), wherein the nucleic acid capture domain of the first probe molecule comprises a sequence with complementarity to first target nucleic acid and the nucleic acid capture domain of the second probe molecule comprises a sequence with complementarity to a second target nucleic acid, and wherein the polymer label of the first probe molecule is different from the polymer label of the second probe molecule and provide for independent detection of the first and second target nucleic acids in a nanopore system. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 15, 16, 17, 18, 35, 36)
-
-
10-14. -14. (canceled)
-
19. A method for detecting at least two distinct single stranded target nucleic acids in a sample with a nanopore system, the method comprising:
-
a) contacting the sample with a set of at least two probe molecules and allowing the probe molecules to hybridize with any target nucleic acids present in the sample to form a hybridized sample, wherein the set of probe molecules comprises at least a first probe molecule and a second probe molecule, both of which comprise; (i) a capture domain that comprises a sequence with complementarity to a target nucleic acid; (ii) a terminal extension that is covalently linked to the 5′
end 3′
end, or both the 5′
end and the 3′
end of the capture domain; and(ii) at least one polymer label attached to at least one of the terminal extension(s), wherein the nucleic acid capture domain of the first probe molecule hybridizes with a first target nucleic acid and the nucleic acid capture domain of the second probe molecule hybridizes with a second target nucleic acid, and wherein the polymer label of the first probe molecule is different from the polymer label of the second probe molecule; b) applying a voltage to said hybridized sample mixture in a cis compartment of a dual chamber nanopore system sufficient to trap a hybridized probe/target nucleic acid complex in the nanopore and drive translocation of said hybridized probes and target nucleic acids through a nanopore of said system by an unzipping process, and, c) analyzing an electrical current pattern in said nanopore system over time, wherein presence of said distinct single stranded target nucleic acids in the sample is indicated by occurrence of two distinct signature electrical current blocks corresponding to trapping of each distinct hybridized probe and target nucleic acids in the nanopore. - View Dependent Claims (20, 34)
-
-
21-33. -33. (canceled)
-
37. A probe molecule comprising:
-
(i) a capture domain that comprises a sequence with complementarity to a target nucleic acid; (ii) a terminal extension that is covalently linked to the 5′
end, the 3′
end, or both the 5′ and
3′
end of the capture domain, and(ii) at least one polymer label attached to at least one terminal extension, wherein said probe molecule provides for detection of the target nucleic acid in a nanopore system. - View Dependent Claims (52)
-
-
38-51. -51. (canceled)
Specification