Nucleic acid detection and quantification by post-hybridization labeling and universal encoding
The present invention provides, among other things, methods and compositions for encoding a substrate for detecting and quantifying target nucleic acids.
1. A method of encoding a particle comprising:
a. providing a particle comprising two or more encoding regions, each encoding region bearing one or more anchor oligonucleotides and wherein the encoding regions are separated by an inert region;
b. providing 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;
c. incubating the particle 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, thereby encoding the substrate by the desired detectable signal level in the each encoding region.
2. The method of claim 1 , wherein the method further comprises a step of coupling the individual encoding adapter to its corresponding anchor oligonucleotide.
3. The method of claim 1 , wherein the anchor oligonucleotide is a single-stranded polynucleotide template.
4. The method of claim 3 , wherein individual single-stranded polynucleotide template and individual encoding adapter are both linear.
5. The method of claim 3 , 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.
6. The method of claim 1 , wherein the labeled encoding adapters are labeled with distinct detectable moieties.
7. The method 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.
8. The method of claim 1 , wherein the particle further comprises one or more probe regions that are distinct from the one or more encoding regions.
9. The method of claim 1 , wherein the particle is a hydrogel particle.
10. The method of claim 1 , wherein the individual anchor oligonucleotide comprises at least 10 bases.
11. The method of claim 1 , wherein the each individual encoding adapter is DNA or RNA.
12. The method of claim 1 , wherein the each individual encoding adapter comprises up to 30 nucleotides.
13. The method of claim 1 , wherein the detectable moiety is selected from fluorophores, chromophores, radioisotopes, quantum dots, nanoparticles and/or intercalating DNA/RNA dyes.
14. The method of claim 1 , wherein a signal provided by at least one labeled encoding adapter is used to determine the orientation of the substrate.
15. The method of claim 1 , wherein a signal provided by at least one labeled encoding adapter is used to normalized the desired detectable signal level.
16. The method of claim 1 , wherein the desired detectable signal level in the each encoding region is distinguishable at multiple levels.
17. The method of claim 16 , wherein the desired detectable signal level in the each encoding region is distinguishable for up to 10 levels.
18. The method of claim 16 , wherein the desired detectable signal level in the each encoding region is distinguishable for up to 20 levels.
19. The method of claim 2 , wherein the step of coupling comprises enzymatic coupling or chemical coupling.
20. The method of claim 18 , wherein the enzymatic couple is ligation.
21. The method of claim 5 , wherein the method further comprises, after the incubating step, a step of ligation such that the individual single-stranded polynucleotide template is covalently coupled with the individual single-stranded encoding adapter bound to the template.
22. A method of encoding a hydrogel particle, comprising:
a. providing a hydrogel particle comprising two or more encoding regions, each encoding region bearing one or more anchor oligonucleotides, wherein each individual anchor oligonucleotide is a single-stranded polynucleotide template that forms a hairpin structure and wherein the encoding regions are separated by an inert region;
b. providing 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;
c. incubating the hydrogel particle 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, thereby encoding the substrate by generating the desired detectable signal level in the each encoding region.