On-slide staining by primer extension
A method for analyzing planar sample is provided. In some cases the method comprises: (a) incubating the planar sample with a capture agent that is linked to an oligonucleotide, 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 oligonucleotide, using fluorescence microscopy. Several implementations of the method, and multiplexed versions of the same, are also provided.
1. A composition comprising:
(a) a plurality of capture agents, wherein the plurality of capture agents can specifically bind to different complementary sites in a biological sample,
wherein each of the plurality of capture agents comprises a double stranded nucleic acid, and wherein a 3′ end of a first strand of the double stranded nucleic acid is recessed relative to a 5′ end of a second strand of the double stranded nucleic acid, thereby producing a 5′ overhang;
(b) a polymerase;
(c) a plurality of nucleotides;
wherein the polymerase is configured to extend a strand of the double stranded nucleic acid with the plurality of nucleotides; and
wherein:
(i) the plurality of nucleotides comprise a fluorescent nucleotide and/or
(ii) the composition further comprises a fluorescently labeled oligonucleotide complementary to a sequence of an overhang of at least one capture agent of the plurality of capture agents.
2. The composition of claim 1 , wherein the plurality of nucleotides comprises the fluorescent nucleotide.
3. The composition of claim 1 , wherein all nucleotides of the plurality of nucleotides are fluorescent.
4. The composition of claim 1 , wherein the plurality of nucleotides is an incomplete nucleotide mix.
5. The composition of claim 1 , wherein the plurality of nucleotides comprises reversible terminator nucleotides or fluorescent reversible terminator nucleotides.
6. The composition of claim 1 , comprising the fluorescently labeled oligonucleotide complementary to the sequence of the overhang of at least one capture agent of the plurality of capture agents.
7. The composition of claim 6 , wherein the fluorescently labeled oligonucleotide comprises a quencher.
8. The composition of claim 7 , wherein the quencher is configured to be removed upon extension of a strand of the double stranded nucleic acid with the plurality of nucleotides.
9. The composition of claim 1 , wherein an overhang of the double stranded nucleic acid of a first capture agent of the plurality of capture agents comprises a different sequence than an overhang of a second capture agent.
10. The composition of the claim 1 , wherein the plurality of capture agents comprises an antibody, aptamer, or oligonucleotide probe.
11. The composition of claim 1 , wherein the different complementary sites comprise protein epitopes or complementary nucleic acids.
12. The composition of claim 1 , wherein a sequence of the first strand is the same for each capture agent of the plurality of capture agents.
13. The composition of claim 1 , wherein the plurality of capture agents comprises at least 5 capture agents.
14. The composition of claim 1 , wherein the plurality of capture agents comprises at least 10 capture agents.