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 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 is 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 is chemically cross-linked directly to the biological sample.
2. The composition of claim 1 , wherein the plurality of capture agents is cross-linked to the biological sample via a bifunctional cross-linker.
3. The composition of claim 1 , wherein the plurality of capture agents is cross-linked to the biological sample via an amine-to-amine crosslinker.
4. The composition of claim 1 , wherein the plurality of capture agents is cross-linked to the biological sample by formaldehyde.
5. The composition of claim 1 , wherein the plurality of capture agents is cross-linked to the biological sample by a disuccinimidyl crosslinker.
6. The composition of claim 1 , wherein the biological sample is planar.
7. The composition of claim 1 , wherein the biological sample is a tissue section.
8. The composition of claim 1 , wherein the biological sample is a formalin-fixed paraffin embedded (FFPE) tissue section.
9. The composition of claim 1 , wherein the biological sample is a tissue biopsy.
10. The composition of claim 1 , wherein the plurality of capture agents is bound to diagnostic markers in the biological sample.
11. The composition 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.
12. The composition of claim 1 , wherein the plurality of capture agents comprises at least 10 capture agents, each linked to a different double-stranded oligonucleotide.
13. The composition of claim 1 , wherein the plurality of capture agents comprises at least 50 capture agents, each linked to a different double-stranded oligonucleotide.
14. The composition of claim 1 , wherein the double-stranded oligonucleotide comprises an overhang.
15. The composition of claim 1 , wherein the plurality of capture agents are antibodies.
16. The composition of claim 1 , wherein the plurality of capture agents are aptamers or oligonucleotide probes.
17. The composition of claim 15 , wherein the antibodies are monoclonal antibodies.
18. The composition of claim 1 , wherein the double-stranded oligonucleotides linked to the plurality of capture agents are at least 10 nucleotides in length.
19. The composition of claim 1 , wherein the double-stranded oligonucleotides linked to the plurality of capture agents are in the range of 15-200 nucleotides in length.
20. The composition of claim 1 , wherein the labeled oligonucleotide comprises a fluorescent label.