Methods for detection and quantification of analytes in complex mixtures

• US 7,919,237 B2
• Filed: 07/03/2002
• Issued: 04/05/2011
• Est. Priority Date: 07/03/2001
• Status: Expired due to Fees

• Current Assignee: Institute for System Biology
• Original Assignee: Nanostring Technologies, Inc.
• Inventors: Dimitrov, Krassen, Dunaway, Dwayne
• Primary Examiner(s): Chunduru; Suryaprabha
• Application Number:
  US10/542,458
• Publication Number:
  US 20070166708A1
• Time in Patent Office: 3,198 Days
• Field of Search: 435/6, 536/24.31, 536/24.32
• US Class Current: 435/6.1
• 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 are methods of using and producing these populations of uniquely labeled nucleic acid probes.

Claims

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 an electro-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.