Super-resolution sequencing

1. A method for template-directed sequencing-by-synthesis of an array of target polynucleotides, the method comprising:

(a) providing an array of target polynucleotides in a fluidic vessel;

(b) contacting the array of polynucleotides with a solution comprising (i) polymerization complex and (ii) reversibly terminating and differently labeled A,C,G, and T/U nucleotides;

(c) incorporating one of the differently labeled nucleotides, using the polymerization complex, into a chain complementary to at least one of the array of polynucleotides;

(d) binding imaging tags to the differently labeled nucleotides of step (c);

(e) imaging by single molecule localization and/or stochastic optical reconstruction and storing the identity and position of the imaging tags of step (d);

(f) reversing termination (b)-(e);

(g) repeating steps (b)-(e) and assembling a sequence for each of the array of target polynucleotides from the stored identity and position of the imaging tags, optionally as a homogeneous or one pot reaction.

2. The method of claim 1 , wherein binding the tags comprises multiple, stochastic on/off binding events.

3. The method of claim 1 , wherein the target polynucleotide is disposed on a surface.

4. The method of claim 1 , wherein the target polynucleotide is stretched and/or elongated.

5. The method of claim 4 , further comprising seeding the incorporation at multiple locations on the stretched and/or elongated target polynucleotide.

6. The method of claim 1 , wherein the incorporation comprises extension from a nick, extension from an oligonucleotide, use of a DNA polymerase not requiring a primer, or transcription from a promoter.

7. The method of claim 1 , wherein an intercalating dye or a fluorescent/luminescent entity is provided as a RET donor and the label on the nucleotide is a RET acceptor.

8. The method of claim 1 , further comprising sequencing two or more polynucleotides simultaneously.

9. The method of claim 1 , wherein the labeled nucleotide comprises a fluorescent organic dye or fluorescent nanoparticle.

10. The method of claim 1 , wherein the labeled nucleotide comprises a quencher.

11. The method of claim 1 , wherein the label comprises a first partner of a binding pair.

12. The method of claim 11 , wherein the second partner of a binding pair comprises a fluorescently labeled transiently binding oligonucleotide.

13. The method of claim 1 , wherein the label is superresolved.

14. The method of claim 1 , wherein the imaging or illuminating step further comprises providing electromagnetic radiation via an evanescent wave.

15. The method of claim 1 , wherein the imaging or illuminating step further comprises enhancing the fluorescence by proximity related effects with metals.

16. The method of claim 1 , wherein the imaging or illuminating step further comprises controlling the attraction and repulsion of the labeled nucleotides using an electric field.

17. The method of claim 1 , wherein assembling the sequence comprises single molecule localization, stochastic optical reconstruction microscopy (STORM), points accumulation for imaging in nanoscale topography (PAINT), or stimulated emission depletion (STED).