On-slide staining by primer extension

1. A method for analyzing a planar sample, the method comprising:

(a) incubating the planar sample with a capture agent under conditions by which the capture agent specifically binds to complementary sites in the planar sample, wherein:

(i) the capture agent is linked to a double-stranded oligonucleotide that comprises a first strand and a second strand;

(ii) the capture agent is linked to a double-stranded oligonucleotide by the 5′ end of the first strand; and

(iii) the 3′ end of the first strand is recessed relative to the 5′ end of the second strand, thereby producing an overhang;

(b) treating the planar sample with a chemical crosslinker to crosslink the capture agent directly to the planar sample;

(c) contacting the planar sample with a polymerase and a nucleotide mix, thereby adding one or more nucleotides to the overhang; and

(d) reading a fluorescent signal generated by addition of the one or more nucleotides to the overhang using fluorescence microscopy, thereby producing an image showing the pattern of binding of the capture agent to the planar sample.

2. The method of claim 1 , wherein:

step (c) comprises contacting the planar sample with a polymerase and a nucleotide mix that comprises a fluorescent nucleotide, thereby adding the fluorescent nucleotide to the overhang; and

step (d) comprises reading a fluorescent signal generated by addition of the fluorescent nucleotide to the overhang.

3. The method of claim 2 , wherein the fluorescent signal is emitted directly from the added nucleotide.

4. The method of claim 2 , wherein the fluorescent signal is a FRET signal generated by energy transfer between two fluorescent nucleotides that are added to the overhang.

5. The method of claim 2 , wherein the fluorescent signal is a FRET signal generated by energy transfer between a first fluorescent nucleotide added to overhang and a second fluorescent nucleotide that is present in the second strand.

6. The method of claim 1 , wherein extension of the first strand removes a quencher from a quenched fluorescently labeled oligonucleotide that is hybridized to the second strand, downstream from the first strand.

7. The method of any prior claim, wherein the sample is a formalin-fixed, paraffin-embedded (FFPE) section.

8. A method for analyzing a tissue sample comprising:

(a) incubating a planar sample with a capture agent composition comprising a plurality of capture agents that recognize different complementary sites, wherein:

(i) each of the capture agents is linked to a double-stranded oligonucleotide that comprises a first strand and a second strand;

(ii) the capture agents are linked to a double-stranded oligonucleotide by the 5′ end of first strand;

(iii) the 3′ end of the first strand in each of the double-stranded oligonucleotides is recessed relative to the 5′ end of the second strand, thereby producing an overhang; and

(iv) the overhang is different for each of the capture agents;

under conditions by which the capture agents specifically bind to sites in the planar sample;

(b) treating the planar sample with a chemical crosslinker to crosslink the capture agents directly to the planar sample;

(c) contacting the planar sample with a polymerase and either an incomplete nucleotide mix or a nucleotide mix that comprises a reversible terminator nucleotide; and

(d) reading, using fluorescence microscopy, a fluorescent signal generated by addition a nucleotide to some but not all of the capture agents.

9. The method of claim 8 , wherein the overhangs are of the formula 3′-N 4n N 1 /N 2 /N 3 , wherein N 1 , N 2 , N 3 and N 4 are different nucleotides selected from G, A, T and C and n is 1 or more.

10. The method of claim 8 , wherein the overhangs are of the formula 3′-YN 1 /N 2 -5′, optionally followed by short stretch of random nucleotides on the 5′ end to increase the overall polymerase residence on the DNA duplex, wherein Y is composed of alternating stretches of bases N 3 and N 4 , N 1 , N 2 are on the same strand, and wherein N 1 , N 2 , N 3 and N 4 are different nucleotides selected from G, A, T and C.

11. The method of claim 8 , wherein:

the sequence of the first strand is the same for each of the capture agents; and

the sequence of the second strand is different for each of the capture agents.

12. The method of claim 8 , wherein the method comprises:

(c) contacting the planar sample with a polymerase and: (i) a nucleotide mix that comprises fluorescent nucleotides that are complementary to N 1 , N 2 and N 3 and a reversible terminator nucleotide that is complementary to N 4 or (ii) a nucleotide mix that comprises fluorescent nucleotides that are complementary to N 1 , and N 2 , an unlabeled nucleotide that is complementary to N 3 , and no nucleotide that is complementary to N 4 , thereby adding fluorescent nucleotides onto the double-stranded oligonucleotides of some but not all of the capture agents, wherein N 1 , N 2 , N 3 and N 4 are different nucleotides selected from G, A, T and C; and

(d) reading, using fluorescence microscopy, a fluorescent signal generated by addition of a fluorescent nucleotide to some but not all of the capture agents.

13. The method of claim 12 , wherein the overhangs are of the formula 3′-N 4n N 1 /N 2 /N 3 , wherein N 1 , N 2 , N 3 and N 4 are different nucleotides selected from G, A, T and C and n is 1 or more; and

step (c) comprises contacting the planar sample with a polymerase and a nucleotide mix that comprises fluorescent nucleotides that are complementary to N 1 , N 2 and N 3 and a reversible terminator nucleotide that is complementary to N 4 .

14. The method of claim 12 , further comprising:

(e) inactivating the fluorescent signal, deprotecting the reversible terminator nucleotide and blocking the sample; and

(f) repeating steps (c) and (d).

15. The method of claim 13 , wherein step (f) comprises repeating steps (c), (d) and (e) multiple times.

16. The method of claim 12 , wherein the overhangs are of the formula 3′-YN 1 /N 2 -5′, optionally followed by short stretch of random nucleotides on the 5′ end to increase the overall polymerase residence on the DNA duplex, wherein Y is composed of alternating stretches of bases N 3 and N 4 , N 1 , N 2 are on the same strand, and wherein N 1 , N 2 , N 3 and N 4 are different nucleotides selected from G, A, T and C.

17. The method of claim 16 , further comprising:

(e) inactivating the fluorescent signal and contacting the planar sample with a polymerase and an unlabeled nucleotide that is complementary to N 4 ; and

(f) repeating steps (c) and (d).

18. The method of claim 17 , wherein step (f) comprises repeating steps (c), (d) and (e) multiple times.

19. The method of claim 8 , wherein the double-stranded oligonucleotides each comprise a fluorescently labeled oligonucleotide hybridized to the second strand downstream from first strand, wherein the fluorescently labeled oligonucleotide comprises a quencher and extension of the first strand removes the quencher from some but not all of the quenched fluorescently labeled oligonucleotides, thereby generating a fluorescent signal for some but not all of the capture agents.

20. The method of claim 8 , wherein:

the sequence of the first strand is the same for each of the capture agents; and

the sequence of the second strand is different for each of the capture agents.

21. The method of claim 8 , wherein the overhangs are of the formula 3′-N 4 n N 1 /N 2 /N 3 , wherein N 1 , N 2 , N 3 and N 4 are different nucleotides selected from G, A, T and C and n is 1 or more.

22. The method of claim 8 , wherein the overhangs are of the formula 3′-YN 1 /N 2 -5′, optionally followed by short stretch of random nucleotides on the 5′ end to increase the overall polymerase residence on the DNA duplex, wherein Y is composed of alternating stretches of bases N 3 and N 4 , N 1 , N 2 are on the same strand, and wherein N 1 , N 2 , N 3 and N 4 are different nucleotides selected from G, A, T and C.