Methods for detection and quantification of analytes in complex mixtures

• US 9,920,380 B2
• Filed: 03/11/2013
• Issued: 03/20/2018
• Est. Priority Date: 07/03/2001
• Status: Expired due to Term

• Current Assignee: Institute for System Biology
• Original Assignee: Institute for System Biology
• Inventors: Dimitrov, Krassen M., Dunaway, Dwayne L.
• Primary Examiner(s): Chunduru, Prabha
• Application Number:
  US13/794,299
• Publication Number:
  US 20140162251A1
• Time in Patent Office: 1,835 Days
• Field of Search: None
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/682 Signal amplification
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 1/6888 for detection or identification of organisms
• CPC Class Codes: C12Q 2525/101 incorporating non-naturally occurring nucleotides, e.g. inosine
• CPC Class Codes: C12Q 2525/151 repeat or repeated sequences, e.g. VNTR, microsatellite, concatemer
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid
• CPC Class Codes: C12Q 2565/102 Multiple non-interacting labels
• CPC Class Codes: C12Q 2565/1025 labels being on the same oligonucleotide

Abstract

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.

Claims

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.