On-slide staining by primer extension
A method for analyzing planar sample is provided. In some cases the method comprises: (a) labelling the planar sample with a capture agent that is linked to a nucleic acid, wherein the capture agent specifically binds to complementary sites in the planar sample; (b) reading a fluorescent signal caused by extension of a primer that is hybridized to the nucleic acid, using fluorescence microscopy. Several implementations of the method, and multiplexed versions of the same, are also provided.
1. A method of analyzing a planar sample, the method comprising:
obtaining a labeled composition comprising:
(a) a biological sample;
(b) a plurality of capture agents;
(c) a labeled oligonucleotide; and
(d) a ligase;
wherein, (i) the plurality of capture agents are linked to double-stranded oligonucleotides comprising a first strand and a second strand, wherein the ligase is configured to add the labeled oligonucleotide to the first strand or the second strand, and (ii) the plurality of capture agents are chemically cross-linked directly to the biological sample; and
reading a signal generated by the labeled composition.
2. The method of claim 1 , wherein the plurality of capture agents are cross-linked to the biological sample via a bifunctional cross-linker.
3. The method of claim 1 , wherein the plurality of capture agents are cross-linked to the biological sample via an amine-to-amine crosslinker.
4. The method of claim 1 , wherein the plurality of capture agents are cross-linked to the biological sample by formaldehyde or a disuccinimidyl crosslinker.
5. The method of claim 1 , wherein the biological sample is planar.
6. The method of claim 1 , wherein the biological sample is a tissue section.
7. The method of claim 1 , wherein the biological sample is a tissue biopsy.
8. The method of claim 1 , wherein the biological sample is a formalin-fixed paraffin embedded tissue section.
9. The method of claim 1 , wherein the plurality of capture agents comprises a first capture agent linked to a first double-stranded oligonucleotide and a second capture agent linked to a second double-stranded oligonucleotide.
10. The method of claim 1 , wherein the plurality of capture agents comprises at least 10 capture agents, each linked to a different double-stranded oligonucleotide.
11. The method of claim 1 , wherein the double-stranded oligonucleotides linked to the plurality of capture agents are at least 10 nucleotides in length.
12. The method of claim 1 , wherein the plurality of capture agents are antibodies.
13. The method of claim 12 , wherein the antibodies are monoclonal antibodies.
14. The method of claim 1 , wherein the plurality of capture agents are aptamers or oligonucleotide probes.
15. The method of claim 1 , wherein the double-stranded oligonucleotides comprises an overhang.
16. The method of claim 1 , wherein the labeled oligonucleotide comprises a fluorescent label.
17. The method of claim 1 , wherein at least one of the plurality of capture agents comprises a fluorescent label.
18. The method of claim 1 , wherein the signal is a fluorescent signal.
19. The method of claim 1 , wherein reading comprises fluorescence microscopy.
20. The method of claim 1 , wherein the method further comprises producing an image showing the pattern of binding of the plurality of capture agents chemically cross-linked directly to the biological sample.