Methods for detection and quantification of analytes in complex mixtures
View Patent ↗The invention provides a method of detecting a nucleic acid analyte. The method consists of (a) contacting a mixture of nucleic acid analytes under conditions sufficient for hybridization with a plurality of target specific nucleic acid probes each having a different specifier; (b) contacting the mixture under conditions sufficient for hybridization with a corresponding plurality of antigenedigits each having a unique label, the plurality of anti-genedigits having a diversity sufficient to uniquely hybridize to genedigits within the specifiers, and (c) uniquely detecting a hybridized complex between one or more analytes in the mixture, a target specific probe, and an anti-genedigit.
1. A method of detecting a target nucleic acid analyte, comprising:
(a) contacting a mixture of nucleic acid analytes comprising one or more target nucleic acid analytes with a plurality of target specific probes,
wherein each target specific probe comprises (i) a target specific region that binds to one target nucleic acid analyte and (ii) a region to which is attached a plurality of label monomers that create a unique label for each target specific probe, said label having a detectable signal that distinguishes one target specific probe which binds to a first target nucleic acid from another target specific probe that binds to a different second target nucleic acid,
further wherein the contacting is made under conditions sufficient for hybridization of at least one target specific probe to at least one target nucleic acid analyte,
(b) stretching said at least one nucleic acid analyte hybridized to said target specific probe using a flow-stretch technique, thereby spatially separating said label monomers on at least one said target specific probe, and
(c) detecting the position or order of the spatially separated label monomers in the unique label attached to the at least one target specific probe that is hybridized to a target nucleic acid analyte, wherein said detected unique label for each target specific probe uniquely identifies each target specific analyte, thereby detecting the one or more target nucleic acid analytes.
2. The method of claim 1 , wherein each of said label monomers are selected from the group consisting of a fluorochrome moiety, a fluorescent moiety, a dye moiety or a chemiluminescent moiety.
3. The method of claim 2 , wherein each of said label monomers comprise a fluorescent moiety.
4. The method of any one of claims 1 - 3 wherein the unique label for each target specific probe is composed of 4 label monomers.
5. The method of any one of claims 1 - 3 wherein the unique label for each target specific probe is composed of 5 label monomers.
6. The method of any one of claims 1 - 3 wherein each of said label monomers is 2000 to 3000 bases pairs apart.
7. The method of any one of claims 1 - 3 wherein unbound target specific probes are removed prior to step (c).
8. The method of any one of claims 1 - 3 , wherein a single copy of a target nucleic acid analyte is detected.
9. The method of any one of claims 1 - 3 , wherein said plurality of probes comprises at least one probe for each nucleic acid analyte in said mixture.
10. The method of any one of claims 1 - 3 , wherein said mixture of nucleic acid analytes comprises genomic DNA, cDNA or mRNA.