Methods for detection and quantification of analytes in complex mixtures
View Patent ↗The invention provides a diverse population of uniquely labeled probes, containing about thirty or more target specific nucleic acid probes each attached to a unique label bound to a nucleic acid. Also provided is a method of producing a population of uniquely labeled nucleic acid probes. Also provided is 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 anti-genedigits 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 composition comprising:
(a) a first probe comprising
(i) a region that selectively binds to a first target specific sequence of a nucleic acid analyte, and
(ii) a region comprising a plurality of label-attachment positions linked together, each label-attachment position being of a predetermined nucleotide sequence, wherein at least two of the label-attachment positions comprise a repeated core element, and wherein
a first label-attachment position is attached to a complementary nucleic acid sequence comprising a first plurality of label monomers, wherein the first plurality of label monomers comprises a first label; and
at least a second label-attachment position is attached to at least a second complementary nucleic acid sequence comprising at least a second plurality of label monomers, wherein the second plurality of label monomers comprises at least a second label;
wherein the first label and the second label are spatially-separable for resolution of the first label from the second label; and
(b) a second probe comprising
(i) a region that selectively binds to a second target specific sequence of the nucleic acid analyte, wherein the first target-specific sequence and the second target-specific sequence bind to different regions of the same nucleic acid analyte, and
(ii) an affinity tag.
2. The composition of claim 1 , wherein the first probe further comprises an affinity tag.
3. The composition of claim 1 , wherein the affinity tag is a biotin tag.
4. The composition of claim 2 , wherein the affinity tag is a biotin tag.
5. The composition of claim 1 , wherein the complementary nucleic acid sequence is DNA.
6. The composition of claim 1 , wherein the complementary nucleic acid sequence is RNA.
7. The composition of claim 1 , wherein the complementary nucleic acid sequence is a dendrimer.
8. The composition of claim 1 , wherein the plurality of label-attachment positions comprises at least four label-attachment positions.
9. The composition of claim 1 , wherein the plurality of label-attachment positions are in a predetermined order within the probe.
10. The composition of claim 1 , wherein at least two of the label-attachment positions have different nucleotide sequences.
11. The composition of claim 1 , wherein each label-attachment position has a different nucleotide sequence.
12. The composition of claim 11 , wherein the nucleotide sequence of each label-attachment position differs by at least two nucleotides.
13. The composition of claim 1 , wherein the label-attachment positions are directly linked.
14. The composition of claim 1 , wherein the label-attachment positions are linked via intervening or adapting sequences.
15. The composition of claim 1 , wherein each label monomer comprises a fluorescent moiety.
16. The composition of claim 1 , wherein the affinity tag is a digoxigenin tag.
17. The composition of claim 2 , wherein the affinity tag is a digoxigenin tag.