Nucleic acid detection and quantification by post-hybridization labeling and universal encoding

• US 9,290,816 B2
• Filed: 11/19/2013
• Issued: 03/22/2016
• Est. Priority Date: 06/07/2010
• Status: Active Grant

• Current Assignee: Firefly BioWorks, Inc. (Abcam Plc)
• Original Assignee: Firefly BioWorks, Inc. (Abcam Plc)
• Inventors: Pregibon, Daniel C., Stoner, Isaac, Windemuth, Andreas, Erps, Timothy
• Primary Examiner(s): FORMAN, BETTY J
• Application Number:
  US14/083,621
• Publication Number:
  US 20150005198A1
• Time in Patent Office: 854 Days
• Field of Search: 435/6.1, 435/287.2
• US Class Current: 1/1
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6825 Nucleic acid detection involving sensors
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 1/6888 for detection or identification of organisms
• CPC Class Codes: C12Q 1/689 for bacteria
• CPC Class Codes: C12Q 1/6895 for plants, fungi or algae
• CPC Class Codes: C12Q 1/70 involving virus or bacteriophage immunoassay for viruses G01N33/56983
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2533/107 Probe or oligonucleotide ligation
• CPC Class Codes: C12Q 2565/626 being a flow cytometer
• CPC Class Codes: G01N 15/14 Optical investigation techniques, e.g. flow cytometry
• CPC Class Codes: G01N 21/47 Scattering, i.e. diffuse reflection G01N21/25, G01N21/41 take precedence G01N21/55 takes precedence
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 33/6803 General methods of protein analysis not limited to specific proteins or families of proteins

Abstract

The present invention provides, among other things, methods and compositions for encoding a substrate for detecting and quantifying target nucleic acids.

Claims

1. A kit comprising:

a particle comprising two or more encoding regions and one or more probe regions that are distinct from the encoding regions,

wherein each encoding region is separated from each probe region or encoding region by an inert region,

wherein each encoding region bears one or more anchor oligonucleotides,

wherein each probe region bears one or more probes of interest; and

a plurality of labeled and unlabeled encoding adapters at a ratio to achieve a desired detectable signal level in each encoding region, wherein each individual encoding adapter is designed to specifically bind an individual anchor oligonucleotide and wherein a labeled encoding adapter comprises a detectable moiety;

wherein when the particle is incubated with the plurality of labeled and unlabeled encoding adapters under conditions that allow the labeled and unlabeled encoding adapters to bind their corresponding anchor oligonucleotides in the two or more encoding regions, the particle is encoded by generating the desired detectable signal level in the each encoding region.

2. The kit of claim 1, wherein the individual anchor oligonucleotide is a single-stranded polynucleotide template.

3. The kit of claim 2, wherein

individual single-stranded polynucleotide template forms a hairpin structure and individual encoding adapter does not form a hairpin structure;

or

individual single-stranded polynucleotide template does not form a hairpin structure and individual single-stranded encoding adapter forms a hairpin structure.

4. The kit of claim 2, wherein the single-stranded polynucleotide template comprises a pre-determined nucleotide sequence.

5. The kit of claim 4, wherein the single-stranded polynucleotide template is linear.

6. The kit of claim 4, wherein the single-stranded polynucleotide template comprises a stem-loop structure and the pre-determined nucleotide sequence is adjacent to the stem-loop structure.

7. The kit of claim 4, wherein the pre-determined nucleotide sequence contains a six-nucleotide sequence that is common among all pre-determined sequences and a four-nucleotide sequence that is unique to only a subset.

8. The kit of claim 4, wherein an individual encoding adapter comprises a nucleotide sequence complementary to the pre-determined nucleotide sequence of a corresponding single-stranded polynucleotide template.

9. The kit of claim 1, wherein the kit further comprises a reagent for enzymatic or chemical coupling of an encoding adapter to an anchor oligonucleotide, once the encoding adapter is captured by the anchor oligonucleotide.

10. The kit of claim 9, wherein the reagent comprises a ligase.

11. The kit of claim 1, wherein the one or more anchors are nucleic acid anchors.

12. The kit of claim 1, wherein the one or more anchors are chemical anchors.

13. The kit of claim 1, wherein the detectable moiety is a fluorophore, chromophore, radioisotope, quantum dot, nanoparticle and/or an intercalating DNA/RNA dye.

14. The kit of claim 1, wherein at least one of the labeled encoding adapters is present at a level suitable for signal normalization for other labeled encoding adapters.

15. The kit of claim 1, wherein at least one of the labeled encoding adapters is present at a level suitable to indicate orientation of the particle.

16. The kit of claim 1, wherein the labeled encoding adapters are labeled with different detectable moieties.

17. The kit of claim 1, wherein the plurality of labeled and unlabeled encoding adapters comprise one or more sets of labeled and unlabeled encoding adapters, wherein, in each set, the labeled and unlabeled encoding adapters have substantially identical sequences.

18. The kit of claim 1, wherein the particle is a hydrogel particle.

19. The kit of claim 1, wherein the individual anchor oligonucleotide comprises at least 10 bases.

20. The kit of claim 1, wherein the each individual encoding adapter is DNA or RNA.

21. The kit of claim 1, wherein the each individual encoding adapter comprises up to 30 nucleotides.

22. The kit of claim 1, wherein the desired detectable signal level in the each encoding region is distinguishable at multiple levels.

23. The kit of claim 22, wherein the desired detectable signal level in the each encoding region is distinguishable for up to 10 levels.

24. The kit of claim 22, wherein the desired detectable signal level in the each encoding region is distinguishable for up to 20 levels.

25. A kit comprising a hydrogel particle, comprising:

a hydrogel particle comprising two or more encoding regions and one or more probe regions that are distinct from the encoding regions,

wherein each encoding region is separated from each probe region or encoding region by an inert region,

wherein each probe region bears one or more probes of interest,

wherein each encoding region bears one or more anchor oligonucleotides,

wherein each individual anchor oligonucleotide is a single-stranded polynucleotide template that forms a hairpin structure; and

a plurality of labeled and unlabeled encoding adapters at a ratio to achieve a desired detectable signal level in each encoding region, wherein each individual encoding adapter is designed to specifically bind an individual anchor oligonucleotide and wherein a labeled encoding adapter comprises a detectable moiety;

wherein when the hydrogel particle is incubated with the plurality of labeled and unlabeled encoding adapters under conditions that allow the labeled and unlabeled encoding adapters to bind their corresponding anchor oligonucleotides in the two or more encoding regions, the hydrogel particle is encoded by generating the desired detectable signal level in the each encoding region.