Methods for detection and quantification of analytes in complex mixtures

• US 8,148,512 B2
• Filed: 06/18/2010
• Issued: 04/03/2012
• 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.
• Primary Examiner(s): Chunduru, Prabha
• Application Number:
  US12/819,101
• Publication Number:
  US 20110003715A1
• Time in Patent Office: 655 Days
• Field of Search: 536/24.31, 536/24.32, 435/6, 435/6.12
• US Class Current: 536/24.31
• 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 target specific nucleic acid probes each attached to a unique label bound to a nucleic acid. 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 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.

Claims

1. A probe comprising:

(a) a region that selectively binds to a target nucleic acid analyte,

(b) a region comprising a plurality of label-attachment positions linked together, each label-attachment position being of a predetermined nucleotide sequence, wherein each label-attachment position is attached to a corresponding predetermined label monomer, wherein each label monomer is spatially-separable for visualization and wherein each label monomer comprises a fluorescent moiety; and

(c) a biotin tag;

wherein said plurality of label-attachment positions is at least four label-attachment positions and at least 4 corresponding label monomers.

2. The probe of claim 1, wherein each label monomer is non-covalently attached to the corresponding label-attachment position.

3. The probe of claim 1, wherein said plurality of label-attachment positions is at least five label-attachment positions and at least 5 corresponding label monomers.

4. The probe of claim 1, wherein the plurality of label-attachment positions are in a predetermined order within the probe.

5. The probe of claim 1, wherein at least two of the label-attachment positions have different nucleotide sequences.

6. The probe of claim 1, wherein each label-attachment position has a different nucleotide sequence.

7. The probe of claim 6, wherein nucleotide sequence of each label-attachment position differs by at least two nucleotides.

8. The probe of claim 1, wherein at least two of the label-attachment positions comprise a repeated core element.

9. The probe of claim 1, wherein the nucleotide sequence of each label-attachment position comprises at least one isocytosine or isoguanine nucleotide.

10. The probe of claim 1, wherein the plurality of label-attachment positions are directly linked.

11. The probe of claim 1, wherein the plurality of label-attachment positions are linked via intervening or adapting sequences.