On-slide staining by primer extension

Abstract

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.

What is claimed is: 
     
         1 . A method for analyzing a planar sample, the method comprising:
 (a) labeling the planar sample with a capture agent to produce a labeled sample, wherein:
 (i) the capture agent is linked to a double-stranded nucleic acid that comprises a first strand and a second strand; and 
 (ii) a 3′ end or 5′ end of either the first strand or the second strand is extendible using the other strand as a template; 
   (b) contacting the labeled sample with i. a polymerase and a plurality of nucleotides and/or ii. a labeled oligonucleotide and a ligase, thereby adding one or more nucleotides of the plurality of nucleotides and/or a labeled oligonucleotide to an end of one of the strands of the double-stranded nucleic acid; and   (c) reading a signal generated by addition of the one or more nucleotides and/or labeled oligonucleotide to one of the first strand or the second strand of the double-stranded nucleic acid.   
     
     
         2 . The method of  claim 1 , wherein the signal is a fluorescent signal. 
     
     
         3 . The method of  claim 2 , wherein reading comprises flourescence microscopy. 
     
     
         4 . The method of any prior claim, further comprising producing an image showing the pattern of binding of the capture agent to the planar sample. 
     
     
         5 . The method of any prior claim, wherein:
 step (b) comprises contacting the labeled sample with a polymerase and a plurality of nucleotides that comprises a fluorescent nucleotide, thereby adding the fluorescent nucleotide to one of the first strand or the second strand of the double-stranded nucleic acid; and   step (c) comprises reading a fluorescent signal generated by addition of the fluorescent nucleotide to one of the first strand or the second strand of the double-stranded nucleic acid.   
     
     
         6 . The method of  claim 5 , wherein the fluorescent signal is: i. emitted directly from the added nucleotide; ii. a FRET signal generated by energy transfer between two fluorescent nucleotides of the plurality of flourescent nucleotides that are added to one of the first strand or second strand of the double-stranded nucleic acid; or iii. a FRET signal generated by energy transfer between the added fluorescent nucleotide and a second fluorescent nucleotide that is present in one of the first strand or second strand double-stranded nucleic acid. 
     
     
         7 . The method of any of  claims 1 - 4 , wherein:
 step (b) comprises contacting the labeled sample with a ligase and a labeled oligonucleotide, thereby adding the labeled oligonucleotide to one of the first strand or second strand of the double-stranded nucleic acid; and   step (c) comprises reading a fluorescent signal generated by addition of the labeled oligonucleotide to one of the first strand or second strand of the double-stranded nucleic acid.   
     
     
         8 . The method of  claim 7 , wherein the fluorescent signal is: i. emitted directly from the added labeled nucleotide; ii. a FRET signal generated by energy transfer between two labeled nucleotides that are added to one of the first strand or second strand of the double-stranded nucleic acid; or iii. a FRET signal generated by energy transfer between the labeled nucleotide added to one of the first strand and second strand of the double-stranded nucleic acid and a second labeled nucleotide that is present in the other strand. 
     
     
         9 . The method of  claim 8 , wherein the labeled nucleotide comprises a fluorescent nucleotide. 
     
     
         10 . The method of  claim 1 , wherein extension of one of the first strand or second strand of the double-stranded nucleic acid removes a quencher from a quenched fluorescently labeled oligonucleotide that is hybridized to the other strand, downstream from the first strand. 
     
     
         11 . The method of any of  claims 1 - 10 , wherein the first strand of the double-stranded nucleic acid is a rolling circle amplification (RCA) product, and the second strand of the double-stranded nucleic acid comprises oligonucleotides that are hybridized to multiple sites in the RCA product. 
     
     
         12 . The method of any of  claims 1 - 10 , wherein the first strand of the double-stranded nucleic acid is a first oligonucleotide, and the second strand of the double-stranded nucleic acid is a second oligonucleotide that is hybridized to the first oligonucleotide. 
     
     
         13 . The method of any of  claims 1 - 12 , wherein the planar sample is a formalin-fixed, paraffin-embedded (FFPE) section. 
     
     
         14 . The method of  claim 1 , wherein the capture agent is an antibody, an aptamer, or an oligonucleotide probe. 
     
     
         15 . A capture agent that is linked to a double-stranded nucleic acid, wherein:
 (i) the double-stranded nucleic acid comprises a first strand and a second strand;   (ii) the capture agent is linked to the first strand; and   (iii) the 5′ end or the 3′ end of either the first strand or the second strand is extendible using the other strand as a template.   
     
     
         16 . A capture agent composition comprising a plurality of capture agents that each recognize different complementary sites, wherein:
 each of the plurality of capture agents is linked to a double-stranded nucleic acid that comprises a first strand and a second strand;   the 5′ end or 3′ end of the first or second strand is extendible using the other strand as a template; and   the templates immediately downstream of the extendible ends are different for each of the plurality of capture agents.   
     
     
         17 . The capture agent composition of  claim 16 , wherein:
 the sequence of the first strand is the same for each of the plurality of capture agents; and   the sequence of the second strand is different for each of the plurality of capture agents.   
     
     
         18 . The composition of  claim 16 , wherein the templates immediately adjacent to the extendible 3′ ends 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. 
     
     
         19 . The composition of  claim 16 , wherein the templates immediately adjacent to the extendible 3′ ends are of the formula 3′-YN 1 /N 2 -5′, optionally followed by a 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 N 3  and N 4 , and wherein N 1 , N 2 , N 3  and N4 are different nucleotides selected from G, A, T and C. 
     
     
         20 . A method for analyzing a planar sample comprising:
 (a) labeling the planar sample with a capture agent composition of any of  claims 16 - 19 ;   (b) contacting the labeled sample with i. a polymerase and either an incomplete nucleotide mix or a nucleotide mix that comprises a reversible terminator nucleotide, thereby adding a nucleotide to the plurality of capture agents; and/or ii. a labeled oligonucleotide and a ligase, thereby adding a labeled oligonucleotide to the plurality of capture agents; and   (c) reading a signal generated by addition of the nucleotide or the labeled oligonucleotide to some but not all of the plurality of capture agents.   
     
     
         21 . The method of  claim 20 , wherein the signal comprises a fluorescent signal. 
     
     
         22 . The method of  claim 21 , wherein the reading comprises fluorescent microscopy. 
     
     
         23 . The method of  claim 20 , comprising:
 (b) contacting the planar sample with a polymerase and: (i) a nucleotide mix that comprises a plurality of 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 a plurality of 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 nucleic acids of some but not all of the plurality of capture agents; and   (c) reading, using fluorescence microscopy, a fluorescent signal generated by addition of the fluorescent nucleotides to the double-stranded nucleic acids of some but not all of the plurality of capture agents.   
     
     
         24 . The method of  claim 23 , wherein the templates immediately adjacent to the extendible 3′ end 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 (b) comprises contacting the planar sample with a polymerase and a nucleotide mix that comprises a plurality of fluorescent nucleotides that are complementary to N 1 , N 2  and N 3  and a reversible terminator nucleotide that is complementary to N 4 . 
     
     
         25 . The method of  claim 23 , further comprising:
 (d) inactivating the fluorescent signal,   (e) optionally, deprotecting the reversible terminator nucleotide;   (f) blocking the sample; and   (g) repeating steps (b) and (c).   
     
     
         26 . The method of  claim 25 , wherein step (g) comprises repeating steps (b)-(f) multiple times. 
     
     
         27 . The method of  claim 23 , wherein the templates immediately adjacent to the extendible 3′ end are of the formula 3′-YN 1 /N 2 -5′, optionally followed by a 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 N 3  and N 4 , and wherein N 1 , N 2 , N 3  and N 4  are different nucleotides selected from G, A, T and C. 
     
     
         28 . The method of  claim 27 , further comprising:
 (d) inactivating the fluorescent signal;   (e) contacting the planar sample with a polymerase and an unlabeled nucleotide that is complementary to N 4 ; and   (f) repeating steps (b) and (c).   
     
     
         29 . The method of  claim 28 , wherein step (f) comprises repeating steps (b)-(e) multiple times. 
     
     
         30 . The method of  claim 20 , wherein the double-stranded nucleic acids each comprise a fluorescently labeled oligonucleotide hybridized to the second strand downstream from the 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 plurality of capture agents. 
     
     
         31 . The method of  claim 20 , wherein extension of the double-stranded nucleic acid comprises contacting the planar sample with a mixture of labeled and unlabeled oligonucleotides and a ligase. 
     
     
         32 . The method of  claim 20 , wherein the plurality of capture agents are selected from the group consisting of: antibodies, aptamers, and oligonucleotide probes. 
     
     
         33 . A kit comprising:
 (a) one or more capture agents, wherein the one or more capture agents can specifically bind to complementary sites in a planar sample.   (b) one or more double-stranded nucleic acids comprising a first strand a second strand,   wherein each of the one or more capture agents is linked to the double-stranded nucleic acid, and wherein a 5′ end or 3′ end of either the first strand or the second strand is extendible using the other strand as a template.   
     
     
         34 . The kit of  claim 33 , further comprising a polymerase. 
     
     
         35 . The kit of  claim 34 , further comprising a nucleotide mix comprising at least one of a fluorescent nucleotide, an unlabeled nucleotide, and a reversible terminator nucleotide. 
     
     
         36 . The kit of  claim 33 , wherein the one or more capture agents is selected from the group consisting of: an antibody, an aptamer and an oligonucleotide probe.