In situ detection of nucleotide variants in high noise samples, and compositions and methods related thereto

Abstract

The invention relates to methods of in situ detection of a nucleic acid variation of a target nucleic acid in a sample, including single nucleotide variations, multi-nucleotide variations or splice sites. The method can comprise the steps of contacting the sample with a probe that detects the nucleic acid variation or splice site and a neighbor probe; contacting the sample with pre-amplifiers that bind to the nucleic acid variation probe or splice site probe and neighbor probe, respectively; contacting the sample with a collaboration amplifier that binds to the pre-amplifiers; and contacting the sample with a label probe system, wherein hybridization of the components forms a signal generating complex (SGC) comprising a target nucleic acid with the nucleic acid variation or splice site, the probes and amplifiers; and detecting in situ signal from the SGC on the sample. The invention also provides samples, tissue slides, and kits relating to detection of nucleic acid variations, including single nucleotide variations, multi-nucleotide variations or splice sites, of a target nucleic acid.

What is claimed is: 
     
         1 . A method of in situ detection of a single nucleotide variation of a target nucleic acid in a sample of fixed and permeabilized cells, comprising:
 (A) contacting the sample with a single nucleotide variation probe (SP) and a neighbor probe (NP), wherein the SP comprises a target anchor segment (SPAT) that can specifically hybridize to a region of the target nucleic acid comprising the single nucleotide variation and a pre-amplifier anchor segment (SPAP), and wherein the NP comprises a target anchor segment (NPAT) that can hybridize to a region of the target nucleic acid adjacent to the binding site of the SP and a pre-amplifier anchor segment (NPAP);   (B) contacting the sample with an SP pre-amplifier (SPM) and an NP pre-amplifier (NPM), wherein the SPM comprises a segment that can bind to the SP and comprises two or more SP collaboration anchors (SPCAs), and wherein the NPM comprises a segment that can bind to the NP and comprises two or more NP collaboration anchors (NPCAs);   (C) contacting the sample with a collaboration amplifier (COM), wherein the COM comprises a first segment complementary to the SPCA, a second segment complementary to the NPCA, and a third segment comprising a plurality of label amplifier anchor segments;   (D) contacting the sample with a label probe system (LPS), wherein the LPS comprises a plurality of label amplifiers (LMs) and a plurality of label probes (LPs), wherein each LM comprises a segment that can bind to a label amplifier anchor segment of the COM and a plurality of label probe anchor segments, wherein each LP comprises a detectable label and a segment that hybridizes to the label probe anchor segment of LM, wherein the aforesaid hybridizations form a signal generating complex (SGC) comprising a target nucleic acid with the single nucleotide variation, an SP, an NP, an SPM, an NPM, a plurality of COMs, a plurality of LMs, and a plurality of LPs; and   (E) detecting in situ signal from the SGC on the sample.   
     
     
         2 . The method of  claim 1 , wherein the target nucleic acid is RNA. 
     
     
         3 . The method of  claim 1  or  2 , wherein the fixed and permeabilized cells are on a tissue slide. 
     
     
         4 . The method of any one of  claims 1 - 3 , wherein the SPAT is 10 to 20 nucleotides in length. 
     
     
         5 . The method of any one of  claims 1 - 4 , wherein the SPAP is 14 to 28 nucleotides in length. 
     
     
         6 . The method of any one of  claims 1 - 5 , wherein the SP optionally comprises a spacer between the SPAT and the SPAP, wherein the spacer is 1 to 10 nucleotides in length. 
     
     
         7 . The method of any one of  claims 1 - 6 , wherein the NPAT is 16 to 30 nucleotides in length. 
     
     
         8 . The method of any one of  claims 1 - 7 , wherein the NPAP is 14 to 28 nucleotides in length. 
     
     
         9 . The method of any one of  claims 1 - 8 , wherein the NP optionally comprises a spacer between the NPAT and the NPAP, wherein the spacer is 1 to 10 nucleotides in length. 
     
     
         10 . The method of any one of  claims 1 - 9 , wherein the SPM is 50 to 500 nucleotides in length. 
     
     
         11 . The method of any one of  claims 1 - 10 , wherein the NPM is 50 to 500 nucleotides in length. 
     
     
         12 . The method of any one of  claims 1 - 11 , wherein the SPCA is 10 to 20 nucleotides in length. 
     
     
         13 . The method of any one of  claims 1 - 12 , wherein the SPM optionally comprises a spacer between the two or more SPCA, wherein the spacer between the SPCA is independently 1 to 10 nucleotides in length. 
     
     
         14 . The method of any one of  claims 1 - 13 , wherein the NPCA is 10 to 20 nucleotides in length. 
     
     
         15 . The method of any one of  claims 1 - 14 , wherein the NPM optionally comprises a spacer between the two or more NPCA, wherein the spacer between the NPCA is independently 1 to 10 nucleotides in length. 
     
     
         16 . The method of any one of  claims 1 - 15 , wherein the COM is 60 to 900 nucleotides in length. 
     
     
         17 . The method of any one of  claims 1 - 16 , wherein the COM optionally comprises a spacer between the first, second and/or third segments, wherein the spacer between the first, second and/or third segments is independently 1 to 10 nucleotides in length. 
     
     
         18 . The method of any one of  claims 1 - 17 , wherein the third segment of the COM optionally comprises a spacer between the plurality of label amplifier anchor segments, wherein the spacer is independently 1 to 10 nucleotides in length. 
     
     
         19 . The method of any one of  claims 1 - 18 , wherein the plurality of COMs bound to the SPM and NPM is in the range of 2 to 20. 
     
     
         20 . The method of any one of  claims 1 - 19 , wherein the plurality of LMs bound to the COM is in the range of 2 to 20. 
     
     
         21 . The method of any one of  claims 1 - 20 , wherein the plurality of LPs bound to the LM is in the range of 2 to 20. 
     
     
         22 . A sample of fixed and permeabilized cells, comprising:
 (A) at least one fixed and permeabilized cell containing a target nucleic acid with a single nucleotide variation;   (B) a single nucleotide variation probe (SP) comprising a target anchor segment (SPAT) hybridized to a region of the target nucleic acid comprising the single nucleotide variation, and, a neighbor probe (NP) comprising a target anchor segment (NPAT) hybridized to a region of the target nucleic acid adjacent to the binding site of the SP;   (C) an SP pre-amplifier (SPM) hybridized to the SP, wherein the SPM comprises a plurality of SP collaboration anchors (SPCAs), and, an NP pre-amplifier (NPM) hybridized to the NP, wherein the NPM comprises a plurality of NP collaboration anchors (NPCAs);   (D) a plurality of collaboration amplifiers (COMs) each hybridized to the SPM and the NPM, wherein each COM comprises a first segment complementary to the SPCA, a second segment complementary to the NPCA, and third segment comprising a plurality of label amplifier anchor segments;   (E) a plurality of label amplifiers (LMs) each hybridized to a label amplifier anchor segment of the COM, wherein the LM comprises a plurality of label probe anchor segments; and   (F) a plurality of label probes (LPs) each hybridized to a label probe anchor segment of the LM, wherein the LP comprises a detectable label;   wherein the aforesaid hybridizations form a signal generating complex (SGC) comprising the target nucleic acid with the single nucleotide variation, an SP, an NP, an SPM, an NPM, a plurality of COMs, a plurality of LMs, and a plurality of LPs, and wherein the SGC provides a signal that is detectable and distinguishable from the background noise.   
     
     
         23 . A tissue slide, comprising:
 (A) a slide having immobilized thereon a plurality of fixed and permeabilized cells comprising at least one fixed and permeabilized cell containing a target nucleic acid with a single nucleotide variation;   (B) a single nucleotide variation probe (SP) comprising a target anchor segment (SPAT) hybridized to a region of the target nucleic acid comprising the single nucleotide variation, and, a neighbor probe (NP) comprising a target anchor segment (NPAT) hybridized to a region of the target nucleic acid adjacent to the binding site of the SP;   (C) an SP pre-amplifier (SPM) hybridized to the SP, wherein the SPM comprises a plurality of SP collaboration anchors (SPCAs), and, an NP pre-amplifier (NPM) hybridized to the NP, wherein the NPM comprises a plurality of NP collaboration anchors (NPCAs);   (D) a plurality of collaboration amplifiers (COMs) each hybridized to the SPM and the NPM, wherein each COM comprises a first segment complementary to the SPCA, a second segment complementary to the NPCA, and a third segment comprising a plurality of label amplifier anchor segments;   (E) a plurality of label amplifiers (LMs) each hybridized to a label amplifier anchor segment of the COM, wherein the LM comprises a plurality of label probe anchor segments; and   (F) a plurality of label probes (LPs) each hybridized to a label probe anchor segment of the LM, wherein the LP comprises a detectable label;   wherein the aforesaid hybridizations form a signal generating complex (SGC) comprising the target nucleic acid with the single nucleotide variation, an SP, an NP, an SPM, an NPM, a plurality of COMs, a plurality of LMs, and a plurality of LPs, and wherein the SGC provides a signal that is detectable and distinguishable from the background noise.   
     
     
         24 . A kit for in situ detection of a single nucleotide variation of a target nucleic acid in a sample of fixed and permeabilized cells, comprising:
 (A) at least one reagent for permeabilizing cells;   (B) a set of target hybridizing probes comprising a single nucleotide variation probe (SP) comprising a target anchor segment (SPAT) capable of hybridizing to a region of the target nucleic acid comprising the single nucleotide variation, and, a neighbor probe (NP) comprising a target anchor segment (NPAT) capable of hybridizing to a region of the target nucleic acid adjacent to the binding site of the SP;   (C) a set of pre-amplifiers comprising an SP pre-amplifier (SPM) comprising a segment capable of hybridizing to the SP, wherein the SPM comprises a plurality of SP collaboration anchors (SPCAs), and, an NP pre-amplifier (NPM) comprising a segment capable of hybridizing to the NP, wherein the NPM comprises a plurality of NP collaboration anchors (NPCAs);   (D) a collaboration amplifier (COM) capable of hybridizing to the SPM and the NPM, wherein the COM comprises a first segment complementary to the SPCA, a second segment complementary to the NPCA, and a third segment comprising a plurality of label amplifier anchor segments;   (E) a label amplifier (LM) capable of hybridizing to the label amplifier anchor segment of the COM, wherein the LM comprises a plurality of label probe anchor segments; and   (F) a label probe (LP) capable of hybridizing to the label probe anchor segment of the LM, wherein the LP comprises a detectable label;   wherein, upon contacting a sample of fixed and permeabilized cells comprising a cell containing a target nucleic acid with the single nucleotide variation, the components in aforesaid (B)-(F) form a signal generating complex (SGC) comprising the target nucleic acid with the single nucleotide variation, an SP, an NP, an SPM, an NPM, a plurality of COMs, a plurality of LMs, and a plurality of LPs, and wherein the SGC provides a signal that is detectable and distinguishable from the background noise.   
     
     
         25 . The sample of  claim 22 , the slide of  claim 23 , or the kit of  claim 24 , wherein the target nucleic acid is RNA. 
     
     
         26 . The sample, slide or kit of any one of  claims 22 - 25 , wherein the fixed and permeabilized cells are on a tissue slide. 
     
     
         27 . The sample, slide or kit of any one of  claims 22 - 26 , wherein the SPAT is 10 to 20 nucleotides in length. 
     
     
         28 . The sample, slide or kit of any one of  claims 22 - 27 , wherein the SPAP is 14 to 28 nucleotides in length. 
     
     
         29 . The sample, slide or kit of any one of  claims 22 - 28 , wherein the SP optionally comprises a spacer between the SPAT and the SPAP, wherein the spacer is 1 to 10 nucleotides in length. 
     
     
         30 . The sample, slide or kit of any one of  claims 22 - 29 , wherein the NPAT is 16 to 30 nucleotides in length. 
     
     
         31 . The sample, slide or kit of any one of  claims 22 - 30 , wherein the NPAP is 14 to 28 nucleotides in length. 
     
     
         32 . The sample, slide or kit of any one of  claims 22 - 31 , wherein the NP optionally comprises a spacer between the NPAT and the NPAP, wherein the spacer is 1 to 10 nucleotides in length. 
     
     
         33 . The sample, slide or kit of any one of  claims 22 - 32 , wherein the SPM is 50 to 500 nucleotides in length. 
     
     
         34 . The sample, slide or kit of any one of  claims 22 - 33 , wherein the NPM is 50 to 500 nucleotides in length. 
     
     
         35 . The sample, slide or kit of any one of  claims 22 - 34 , wherein the SPCA is 10 to 20 nucleotides in length. 
     
     
         36 . The sample, slide or kit of any one of  claims 22 - 35 , wherein the SPM optionally comprises a spacer between the two or more SPCA, wherein the spacer between the SPCA is independently 1 to 10 nucleotides in length. 
     
     
         37 . The sample, slide or kit of any one of  claims 22 - 36 , wherein the NPCA is 10 to 20 nucleotides in length. 
     
     
         38 . The sample, slide or kit of any one of  claims 22 - 37 , wherein the NPM optionally comprises a spacer between the two or more NPCA, wherein the spacer between the NPCA is independently 1 to 10 nucleotides in length. 
     
     
         39 . The sample, slide or kit of any one of  claims 22 - 38 , wherein the COM is 60 to 900 nucleotides in length. 
     
     
         40 . The sample, slide or kit of any one of  claims 22 - 39 , wherein the COM optionally comprises a spacer between the first, second and/or third segments, wherein the spacer between the first, second and/or third segments is independently 1 to 10 nucleotides in length. 
     
     
         41 . The sample, slide or kit of any one of  claims 22 - 40 , wherein the third segment of the COM optionally comprises a spacer between the plurality of label amplifier anchor segments, wherein the spacer is independently 1 to 10 nucleotides in length. 
     
     
         42 . The sample, slide or kit of any one of  claims 22 - 41 , wherein the plurality of COMs bound to the SPM and NPM is in the range of 2 to 20. 
     
     
         43 . The sample, slide or kit of any one of  claims 22 - 42 , wherein the plurality of LMs bound to the COM is in the range of 2 to 20. 
     
     
         44 . The sample, slide or kit of any one of  claims 22 - 43 , wherein the plurality of LPs bound to the LM is in the range of 2 to 20. 
     
     
         45 . A method of in situ detection of a target nucleic acid, wherein the target nucleic acid is 300 or fewer bases in length, in a sample of fixed and permeabilized cells, comprising:
 (A) contacting the sample with a set of target probes (TPs), wherein the set comprises at least two target probes, wherein each TP comprises a target anchor segment (TPAT) that can specifically hybridize to a region of the target nucleic acid and a pre-amplifier anchor segment (TPAP), wherein the set comprises pairs of TPs that can bind to adjacent, non-overlapping regions of the target nucleic acid;   (B) contacting the sample with a set of TP pre-amplifiers (TPMs), wherein the set comprises at least one pair of TPMs, wherein each TPM comprises a segment that can bind to one member of the pair of TPs that bind to adjacent regions of the target nucleic acid, and wherein each TPM comprises two or more TP collaboration anchors (TPCAs);   (C) contacting the sample with a collaboration amplifier (COM), wherein the COM comprises a first segment complementary to the TPCA of one member of the pair of TPMs, a second segment complementary to the TPCA of the second member of the pair of TPMs, and a third segment comprising a plurality of label amplifier anchor segments;   (D) contacting the sample with a label probe system (LPS), wherein the LPS comprises a plurality of label amplifiers (LMs) and a plurality of label probes (LPs), wherein each LM comprises a segment that can bind to a label amplifier anchor segment of the COM and a plurality of label probe anchor segments, wherein each LP comprises a detectable label and a segment that hybridizes to the label probe anchor segment of LM, wherein the aforesaid hybridizations form a signal generating complex (SGC) comprising the target nucleic acid, at least one pair of TPs, at least one pair of TPMs, a plurality of COMs, a plurality of LMs, and a plurality of LPs; and   (E) detecting in situ signal from the SGC on the sample.   
     
     
         46 . A method of in situ detection of a spliced target nucleic acid in a sample of fixed and permeabilized cells, comprising:
 (A) contacting the sample with a splice site probe (SP) and a neighbor probe (NP), wherein the SP comprises a target anchor segment (SPAT) that can specifically hybridize to a region of the target nucleic acid comprising the splice site and a pre-amplifier anchor segment (SPAP), and wherein the NP comprises a target anchor segment (NPAT) that can hybridize to a region of the target nucleic acid adjacent to the binding site of the SP and a pre-amplifier anchor segment (NPAP);   (B) contacting the sample with an SP pre-amplifier (SPM) and an NP pre-amplifier (NPM), wherein the SPM comprises a segment that can bind to the SP and comprises two or more SP collaboration anchors (SPCAs), and wherein the NPM comprises a segment that can bind to the NP and comprises two or more NP collaboration anchors (NPCAs);   (C) contacting the sample with a collaboration amplifier (COM), wherein the COM comprises a first segment complementary to the SPCA, a second segment complementary to the NPCA, and a third segment comprising a plurality of label amplifier anchor segments;   (D) contacting the sample with a label probe system (LPS), wherein the LPS comprises a plurality of label amplifiers (LMs) and a plurality of label probes (LPs), wherein each LM comprises a segment that can bind to a label amplifier anchor segment of the COM and a plurality of label probe anchor segments, wherein each LP comprises a detectable label and a segment that hybridizes to the label probe anchor segment of LM, wherein the aforesaid hybridizations form a signal generating complex (SGC) comprising a target nucleic acid with the splice site, an SP, an NP, an SPM, an NPM, a plurality of COMs, a plurality of LMs, and a plurality of LPs; and   (E) detecting in situ signal from the SGC on the sample.   
     
     
         47 . The method of  claim 46 , wherein the SPAT can specifically hybridize to one of the two spliced nucleic acid segments. 
     
     
         48 . The method of  claim 46 , wherein the SPAT can specifically hybridize to both of the two spliced nucleic acid segments. 
     
     
         49 . The method of any one of  claims 46 - 48 , wherein the target nucleic acid is RNA. 
     
     
         50 . The method of any one of  claims 46 - 49 , wherein the fixed and permeabilized cells are on a tissue slide. 
     
     
         51 . The method of any one of  claims 46 - 50 , wherein the SPAT is 10 to 20 nucleotides in length. 
     
     
         52 . The method of any one of  claims 46 - 51 , wherein the SPAP is 14 to 28 nucleotides in length. 
     
     
         53 . The method of any one of  claims 46 - 52 , wherein the SP optionally comprises a spacer between the SPAT and the SPAP, wherein the spacer is 1 to 10 nucleotides in length. 
     
     
         54 . The method of any one of  claims 46 - 53 , wherein the NPAT is 16 to 30 nucleotides in length. 
     
     
         55 . The method of any one of  claims 46 - 54 , wherein the NPAP is 14 to 28 nucleotides in length. 
     
     
         56 . The method of any one of  claims 46 - 55 , wherein the NP optionally comprises a spacer between the NPAT and the NPAP, wherein the spacer is 1 to 10 nucleotides in length. 
     
     
         57 . The method of any one of  claims 46 - 56 , wherein the SPM is 50 to 500 nucleotides in length. 
     
     
         58 . The method of any one of  claims 46 - 57 , wherein the NPM is 50 to 500 nucleotides in length. 
     
     
         59 . The method of any one of  claims 46 - 58 , wherein the SPCA is 10 to 20 nucleotides in length. 
     
     
         60 . The method of any one of  claims 46 - 59 , wherein the SPM optionally comprises a spacer between the two or more SPCA, wherein the spacer between the SPCA is independently 1 to 10 nucleotides in length. 
     
     
         61 . The method of any one of  claims 46 - 60 , wherein the NPCA is 10 to 20 nucleotides in length. 
     
     
         62 . The method of any one of  claims 46 - 61 , wherein the NPM optionally comprises a spacer between the two or more NPCA, wherein the spacer between the NPCA is independently 1 to 10 nucleotides in length. 
     
     
         63 . The method of any one of  claims 46 - 62 , wherein the COM is 60 to 900 nucleotides in length. 
     
     
         64 . The method of any one of  claims 46 - 63 , wherein the COM optionally comprises a spacer between the first, second and/or third segments, wherein the spacer between the first, second and/or third segments is independently 1 to 10 nucleotides in length. 
     
     
         65 . The method of any one of  claims 46 - 64 , wherein the third segment of the COM optionally comprises a spacer between the plurality of label amplifier anchor segments, wherein the spacer is independently 1 to 10 nucleotides in length. 
     
     
         66 . The method of any one of  claims 46 - 65 , wherein the plurality of COMs bound to the SPM and NPM is in the range of 2 to 20. 
     
     
         67 . The method of any one of  claims 46 - 66 , wherein the plurality of LMs bound to the COM is in the range of 2 to 20. 
     
     
         68 . The method of any one of  claims 46 - 67 , wherein the plurality of LPs bound to the LM is in the range of 2 to 20. 
     
     
         69 . A sample of fixed and permeabilized cells, comprising:
 (A) at least one fixed and permeabilized cell containing a spliced target nucleic acid;   (B) a splice site probe (SP) comprising a target anchor segment (SPAT) hybridized to a region of the target nucleic acid comprising the splice site, and, a neighbor probe (NP) comprising a target anchor segment (NPAT) hybridized to a region of the target nucleic acid adjacent to the binding site of the SP;   (C) an SP pre-amplifier (SPM) hybridized to the SP, wherein the SPM comprises a plurality of SP collaboration anchors (SPCAs), and, an NP pre-amplifier (NPM) hybridized to the NP, wherein the NPM comprises a plurality of NP collaboration anchors (NPCAs);   (D) a plurality of collaboration amplifiers (COMs) each hybridized to the SPM and the NPM, wherein each COM comprises a first segment complementary to the SPCA, a second segment complementary to the NPCA, and third segment comprising a plurality of label amplifier anchor segments;   (E) a plurality of label amplifiers (LMs) each hybridized to a label amplifier anchor segment of the COM, wherein the LM comprises a plurality of label probe anchor segments; and   (F) a plurality of label probes (LPs) each hybridized to a label probe anchor segment of the LM, wherein the LP comprises a detectable label;   wherein the aforesaid hybridizations form a signal generating complex (SGC) comprising the target nucleic acid with the splice site, an SP, an NP, an SPM, an NPM, a plurality of COMs, a plurality of LMs, and a plurality of LPs, and wherein the SGC provides a signal that is detectable and distinguishable from the background noise.   
     
     
         70 . A tissue slide, comprising:
 (A) a slide having immobilized thereon a plurality of fixed and permeabilized cells comprising at least one fixed and permeabilized cell containing a spliced target nucleic acid;   (B) a splice site probe (SP) comprising a target anchor segment (SPAT) hybridized to a region of the target nucleic acid comprising the splice site, and, a neighbor probe (NP) comprising a target anchor segment (NPAT) hybridized to a region of the target nucleic acid adjacent to the binding site of the SP;   (C) an SP pre-amplifier (SPM) hybridized to the SP, wherein the SPM comprises a plurality of SP collaboration anchors (SPCAs), and, an NP pre-amplifier (NPM) hybridized to the NP, wherein the NPM comprises a plurality of NP collaboration anchors (NPCAs);   (D) a plurality of collaboration amplifiers (COMs) each hybridized to the SPM and the NPM, wherein each COM comprises a first segment complementary to the SPCA, a second segment complementary to the NPCA, and a third segment comprising a plurality of label amplifier anchor segments;   (E) a plurality of label amplifiers (LMs) each hybridized to a label amplifier anchor segment of the COM, wherein the LM comprises a plurality of label probe anchor segments; and   (F) a plurality of label probes (LPs) each hybridized to a label probe anchor segment of the LM, wherein the LP comprises a detectable label;   wherein the aforesaid hybridizations form a signal generating complex (SGC) comprising the target nucleic acid with the splice site, an SP, an NP, an SPM, an NPM, a plurality of COMs, a plurality of LMs, and a plurality of LPs, and wherein the SGC provides a signal that is detectable and distinguishable from the background noise.   
     
     
         71 . A kit for in situ detection of a spliced target nucleic acid in a sample of fixed and permeabilized cells, comprising:
 (A) at least one reagent for permeabilizing cells;   (B) a set of target hybridizing probes comprising a splice site probe (SP) comprising a target anchor segment (SPAT) capable of hybridizing to a region of the target nucleic acid comprising the splice site, and, a neighbor probe (NP) comprising a target anchor segment (NPAT) capable of hybridizing to a region of the target nucleic acid adjacent to the binding site of the SP;   (C) a set of pre-amplifiers comprising an SP pre-amplifier (SPM) comprising a segment capable of hybridizing to the SP, wherein the SPM comprises a plurality of SP collaboration anchors (SPCAs), and, an NP pre-amplifier (NPM) comprising a segment capable of hybridizing to the NP, wherein the NPM comprises a plurality of NP collaboration anchors (NPCAs);   (D) a collaboration amplifier (COM) capable of hybridizing to the SPM and the NPM, wherein the COM comprises a first segment complementary to the SPCA, a second segment complementary to the NPCA, and a third segment comprising a plurality of label amplifier anchor segments;   (E) a label amplifier (LM) capable of hybridizing to the label amplifier anchor segment of the COM, wherein the LM comprises a plurality of label probe anchor segments; and   (F) a label probe (LP) capable of hybridizing to the label probe anchor segment of the LM, wherein the LP comprises a detectable label;   wherein, upon contacting a sample of fixed and permeabilized cells comprising a cell containing a target nucleic acid with the splice site, the components in aforesaid (B)-(F) form a signal generating complex (SGC) comprising the target nucleic acid with the splice site, an SP, an NP, an SPM, an NPM, a plurality of COMs, a plurality of LMs, and a plurality of LPs, and wherein the SGC provides a signal that is detectable and distinguishable from the background noise.   
     
     
         72 . The sample of  claim 69 , the slide of  claim 70 , or the kit of  claim 71 , wherein the SPAT can specifically hybridize to one of the two spliced nucleic acid segments. 
     
     
         73 . The sample, slide or kit of any one of  claims 69 - 72 , wherein the SPAT can specifically hybridize to both of the two spliced nucleic acid segments. 
     
     
         74 . The sample, slide or kit of any one of  claims 69 - 73 , wherein the target nucleic acid is RNA. 
     
     
         75 . The sample, slide or kit of any one of  claims 69 - 74 , wherein the fixed and permeabilized cells are on a tissue slide. 
     
     
         76 . The sample, slide or kit of any one of  claims 69 - 75 , wherein the SPAT is 10 to 20 nucleotides in length. 
     
     
         77 . The sample, slide or kit of any one of  claims 69 - 76 , wherein the SPAP is 14 to 28 nucleotides in length. 
     
     
         78 . The sample, slide or kit of any one of  claims 69 - 77 , wherein the SP optionally comprises a spacer between the SPAT and the SPAP, wherein the spacer is 1 to 10 nucleotides in length. 
     
     
         79 . The sample, slide or kit of any one of  claims 69 - 78 , wherein the NPAT is 16 to 30 nucleotides in length. 
     
     
         80 . The sample, slide or kit of any one of  claims 69 - 79 , wherein the NPAP is 14 to 28 nucleotides in length. 
     
     
         81 . The sample, slide or kit of any one of  claims 69 - 80 , wherein the NP optionally comprises a spacer between the NPAT and the NPAP, wherein the spacer is 1 to 10 nucleotides in length. 
     
     
         82 . The sample, slide or kit of any one of  claims 69 - 81 , wherein the SPM is 50 to 500 nucleotides in length. 
     
     
         83 . The sample, slide or kit of any one of  claims 69 - 82 , wherein the NPM is 50 to 500 nucleotides in length. 
     
     
         84 . The sample, slide or kit of any one of  claims 69 - 83 , wherein the SPCA is 10 to 20 nucleotides in length. 
     
     
         85 . The sample, slide or kit of any one of  claims 69 - 84 , wherein the SPM optionally comprises a spacer between the two or more SPCA, wherein the spacer between the SPCA is independently 1 to 10 nucleotides in length. 
     
     
         86 . The sample, slide or kit of any one of  claims 69 - 85 , wherein the NPCA is 10 to 20 nucleotides in length. 
     
     
         87 . The sample, slide or kit of any one of  claims 69 - 86 , wherein the NPM optionally comprises a spacer between the two or more NPCA, wherein the spacer between the NPCA is independently 1 to 10 nucleotides in length. 
     
     
         88 . The sample, slide or kit of any one of  claims 69 - 87 , wherein the COM is 60 to 900 nucleotides in length. 
     
     
         89 . The sample, slide or kit of any one of  claims 69 - 88 , wherein the COM optionally comprises a spacer between the first, second and/or third segments, wherein the spacer between the first, second and/or third segments is independently 1 to 10 nucleotides in length. 
     
     
         90 . The sample, slide or kit of any one of  claims 69 - 89 , wherein the third segment of the COM optionally comprises a spacer between the plurality of label amplifier anchor segments, wherein the spacer is independently 1 to 10 nucleotides in length. 
     
     
         91 . The sample, slide or kit of any one of  claims 69 - 90 , wherein the plurality of COMs bound to the SPM and NPM is in the range of 2 to 20. 
     
     
         92 . The sample, slide or kit of any one of  claims 69 - 91 , wherein the plurality of LMs bound to the COM is in the range of 2 to 20. 
     
     
         93 . The sample, slide or kit of any one of  claims 69 - 92 , wherein the plurality of LPs bound to the LM is in the range of 2 to 20. 
     
     
         94 . A method of in situ detection of a nucleotide variation of a target nucleic acid in a sample of fixed and permeabilized cells, comprising:
 (A) contacting the sample with a nucleotide variation probe (SP) and a neighbor probe (NP), wherein the SP comprises a target anchor segment (SPAT) that can specifically hybridize to a region of the target nucleic acid comprising the nucleotide variation and a pre-amplifier anchor segment (SPAP), and wherein the NP comprises a target anchor segment (NPAT) that can hybridize to a region of the target nucleic acid adjacent to the binding site of the SP and a pre-amplifier anchor segment (NPAP);   (B) contacting the sample with an SP pre-amplifier (SPM) and an NP pre-amplifier (NPM), wherein the SPM comprises a segment that can bind to the SP and comprises two or more SP collaboration anchors (SPCAs), and wherein the NPM comprises a segment that can bind to the NP and comprises two or more NP collaboration anchors (NPCAs);   (C) contacting the sample with a collaboration amplifier (COM), wherein the COM comprises a first segment complementary to the SPCA, a second segment complementary to the NPCA, and a third segment comprising a plurality of label amplifier anchor segments;   (D) contacting the sample with a label probe system (LPS), wherein the LPS comprises a plurality of label amplifiers (LMs) and a plurality of label probes (LPs), wherein each LM comprises a segment that can bind to a label amplifier anchor segment of the COM and a plurality of label probe anchor segments, wherein each LP comprises a detectable label and a segment that hybridizes to the label probe anchor segment of LM, wherein the aforesaid hybridizations form a signal generating complex (SGC) comprising a target nucleic acid with the nucleotide variation, an SP, an NP, an SPM, an NPM, a plurality of COMs, a plurality of LMs, and a plurality of LPs; and   (E) detecting in situ signal from the SGC on the sample.   
     
     
         95 . The method of  claim 94 , wherein the nucleotide variation is selected from the group consisting of a single nucleotide variation, a multi-nucleotide variation, a splice site, an insertion, a deletion, and a rearrangement. 
     
     
         96 . The method of  claim 94  or  95 , wherein the target nucleic acid is RNA. 
     
     
         97 . The method of any one of  claims 94 - 96 , wherein the fixed and permeabilized cells are on a tissue slide. 
     
     
         98 . The method of any one of  claims 1 - 97 , wherein the SPAT is 10 to 20 nucleotides in length. 
     
     
         99 . The method of any one of  claims 1 - 98 , wherein the SPAP is 14 to 28 nucleotides in length. 
     
     
         100 . The method of any one of  claims 1 - 99 , wherein the SP optionally comprises a spacer between the SPAT and the SPAP, wherein the spacer is 1 to 10 nucleotides in length. 
     
     
         101 . The method of any one of  claims 1 - 100 , wherein the NPAT is 16 to 30 nucleotides in length. 
     
     
         102 . The method of any one of  claims 1 - 101 , wherein the NPAP is 14 to 28 nucleotides in length. 
     
     
         103 . The method of any one of  claims 1 - 102 , wherein the NP optionally comprises a spacer between the NPAT and the NPAP, wherein the spacer is 1 to 10 nucleotides in length. 
     
     
         104 . The method of any one of  claims 1 - 103 , wherein the SPM is 50 to 500 nucleotides in length. 
     
     
         105 . The method of any one of  claims 1 - 104 , wherein the NPM is 50 to 500 nucleotides in length. 
     
     
         106 . The method of any one of  claims 1 - 105 , wherein the SPCA is 10 to 20 nucleotides in length. 
     
     
         107 . The method of any one of  claims 1 - 106 , wherein the SPM optionally comprises a spacer between the two or more SPCA, wherein the spacer between the SPCA is independently 1 to 10 nucleotides in length. 
     
     
         108 . The method of any one of  claims 1 - 107 , wherein the NPCA is 10 to 20 nucleotides in length. 
     
     
         109 . The method of any one of  claims 1 - 108 , wherein the NPM optionally comprises a spacer between the two or more NPCA, wherein the spacer between the NPCA is independently 1 to 10 nucleotides in length. 
     
     
         110 . The method of any one of  claims 1 - 109 , wherein the COM is 60 to 900 nucleotides in length. 
     
     
         111 . The method of any one of  claims 1 - 110 , wherein the COM optionally comprises a spacer between the first, second and/or third segments, wherein the spacer between the first, second and/or third segments is independently 1 to 10 nucleotides in length. 
     
     
         112 . The method of any one of  claims 1 - 111 , wherein the third segment of the COM optionally comprises a spacer between the plurality of label amplifier anchor segments, wherein the spacer is independently 1 to 10 nucleotides in length. 
     
     
         113 . The method of any one of  claims 1 - 112 , wherein the plurality of COMs bound to the SPM and NPM is in the range of 2 to 20. 
     
     
         114 . The method of any one of  claims 1 - 113 , wherein the plurality of LMs bound to the COM is in the range of 2 to 20. 
     
     
         115 . The method of any one of  claims 1 - 114 , wherein the plurality of LPs bound to the LM is in the range of 2 to 20. 
     
     
         116 . A sample of fixed and permeabilized cells, comprising:
 (A) at least one fixed and permeabilized cell containing a target nucleic acid with a nucleotide variation;   (B) a nucleotide variation probe (SP) comprising a target anchor segment (SPAT) hybridized to a region of the target nucleic acid comprising the nucleotide variation, and, a neighbor probe (NP) comprising a target anchor segment (NPAT) hybridized to a region of the target nucleic acid adjacent to the binding site of the SP;   (C) an SP pre-amplifier (SPM) hybridized to the SP, wherein the SPM comprises a plurality of SP collaboration anchors (SPCAs), and, an NP pre-amplifier (NPM) hybridized to the NP, wherein the NPM comprises a plurality of NP collaboration anchors (NPCAs);   (D) a plurality of collaboration amplifiers (COMs) each hybridized to the SPM and the NPM, wherein each COM comprises a first segment complementary to the SPCA, a second segment complementary to the NPCA, and third segment comprising a plurality of label amplifier anchor segments;   (E) a plurality of label amplifiers (LMs) each hybridized to a label amplifier anchor segment of the COM, wherein the LM comprises a plurality of label probe anchor segments; and   (F) a plurality of label probes (LPs) each hybridized to a label probe anchor segment of the LM, wherein the LP comprises a detectable label;   wherein the aforesaid hybridizations form a signal generating complex (SGC) comprising the target nucleic acid with the nucleotide variation, an SP, an NP, an SPM, an NPM, a plurality of COMs, a plurality of LMs, and a plurality of LPs, and wherein the SGC provides a signal that is detectable and distinguishable from the background noise.   
     
     
         117 . A tissue slide, comprising:
 (A) a slide having immobilized thereon a plurality of fixed and permeabilized cells comprising at least one fixed and permeabilized cell containing a target nucleic acid with a nucleotide variation;   (B) a nucleotide variation probe (SP) comprising a target anchor segment (SPAT) hybridized to a region of the target nucleic acid comprising the nucleotide variation, and, a neighbor probe (NP) comprising a target anchor segment (NPAT) hybridized to a region of the target nucleic acid adjacent to the binding site of the SP;   (C) an SP pre-amplifier (SPM) hybridized to the SP, wherein the SPM comprises a plurality of SP collaboration anchors (SPCAs), and, an NP pre-amplifier (NPM) hybridized to the NP, wherein the NPM comprises a plurality of NP collaboration anchors (NPCAs);   (D) a plurality of collaboration amplifiers (COMs) each hybridized to the SPM and the NPM, wherein each COM comprises a first segment complementary to the SPCA, a second segment complementary to the NPCA, and a third segment comprising a plurality of label amplifier anchor segments;   (E) a plurality of label amplifiers (LMs) each hybridized to a label amplifier anchor segment of the COM, wherein the LM comprises a plurality of label probe anchor segments; and   (F) a plurality of label probes (LPs) each hybridized to a label probe anchor segment of the LM, wherein the LP comprises a detectable label;   wherein the aforesaid hybridizations form a signal generating complex (SGC) comprising the target nucleic acid with the nucleotide variation, an SP, an NP, an SPM, an NPM, a plurality of COMs, a plurality of LMs, and a plurality of LPs, and wherein the SGC provides a signal that is detectable and distinguishable from the background noise.   
     
     
         118 . A kit for in situ detection of a nucleotide variation of a target nucleic acid in a sample of fixed and permeabilized cells, comprising:
 (A) at least one reagent for permeabilizing cells;   (B) a set of target hybridizing probes comprising a nucleotide variation probe (SP) comprising a target anchor segment (SPAT) capable of hybridizing to a region of the target nucleic acid comprising the nucleotide variation, and, a neighbor probe (NP) comprising a target anchor segment (NPAT) capable of hybridizing to a region of the target nucleic acid adjacent to the binding site of the SP;   (C) a set of pre-amplifiers comprising an SP pre-amplifier (SPM) comprising a segment capable of hybridizing to the SP, wherein the SPM comprises a plurality of SP collaboration anchors (SPCAs), and, an NP pre-amplifier (NPM) comprising a segment capable of hybridizing to the NP, wherein the NPM comprises a plurality of NP collaboration anchors (NPCAs);   (D) a collaboration amplifier (COM) capable of hybridizing to the SPM and the NPM, wherein the COM comprises a first segment complementary to the SPCA, a second segment complementary to the NPCA, and a third segment comprising a plurality of label amplifier anchor segments;   (E) a label amplifier (LM) capable of hybridizing to the label amplifier anchor segment of the COM, wherein the LM comprises a plurality of label probe anchor segments; and   (F) a label probe (LP) capable of hybridizing to the label probe anchor segment of the LM, wherein the LP comprises a detectable label;   wherein, upon contacting a sample of fixed and permeabilized cells comprising a cell containing a target nucleic acid with the nucleotide variation, the components in aforesaid (B)-(F) form a signal generating complex (SGC) comprising the target nucleic acid with the nucleotide variation, an SP, an NP, an SPM, an NPM, a plurality of COMs, a plurality of LMs, and a plurality of LPs, and wherein the SGC provides a signal that is detectable and distinguishable from the background noise.   
     
     
         119 . The sample of  claim 116 , the slide of  claim 117 , or the kit of  claim 118 , wherein the nucleotide variation is selected from the group consisting of a single nucleotide variation, a multi-nucleotide variation, a splice site, an insertion, a deletion, and a rearrangement. 
     
     
         120 . The sample, slide or kit of any one of  claims 116 - 119 , wherein the target nucleic acid is RNA. 
     
     
         121 . The sample, slide or kit of any one of  claims 116 - 120 , wherein the fixed and permeabilized cells are on a tissue slide. 
     
     
         122 . The sample, slide or kit of any one of  claims 116 - 121 , wherein the SPAT is 10 to 20 nucleotides in length. 
     
     
         123 . The sample, slide or kit of any one of  claims 116 - 122 , wherein the SPAP is 14 to 28 nucleotides in length. 
     
     
         124 . The sample, slide or kit of any one of  claims 116 - 123 , wherein the SP optionally comprises a spacer between the SPAT and the SPAP, wherein the spacer is 1 to 10 nucleotides in length. 
     
     
         125 . The sample, slide or kit of any one of  claims 116 - 124 , wherein the NPAT is 16 to 30 nucleotides in length. 
     
     
         126 . The sample, slide or kit of any one of  claims 116 - 125 , wherein the NPAP is 14 to 28 nucleotides in length. 
     
     
         127 . The sample, slide or kit of any one of  claims 116 - 126 , wherein the NP optionally comprises a spacer between the NPAT and the NPAP, wherein the spacer is 1 to 10 nucleotides in length. 
     
     
         128 . The sample, slide or kit of any one of  claims 116 - 127 , wherein the SPM is 50 to 500 nucleotides in length. 
     
     
         129 . The sample, slide or kit of any one of  claims 116 - 128 , wherein the NPM is 50 to 500 nucleotides in length. 
     
     
         130 . The sample, slide or kit of any one of  claims 116 - 129 , wherein the SPCA is 10 to 20 nucleotides in length. 
     
     
         131 . The sample, slide or kit of any one of  claims 116 - 130 , wherein the SPM optionally comprises a spacer between the two or more SPCA, wherein the spacer between the SPCA is independently 1 to 10 nucleotides in length. 
     
     
         132 . The sample, slide or kit of any one of  claims 116 - 131 , wherein the NPCA is 10 to 20 nucleotides in length. 
     
     
         133 . The sample, slide or kit of any one of  claims 116 - 132 , wherein the NPM optionally comprises a spacer between the two or more NPCA, wherein the spacer between the NPCA is independently 1 to 10 nucleotides in length. 
     
     
         134 . The sample, slide or kit of any one of  claims 116 - 133 , wherein the COM is 60 to 900 nucleotides in length. 
     
     
         135 . The sample, slide or kit of any one of  claims 116 - 134 , wherein the COM optionally comprises a spacer between the first, second and/or third segments, wherein the spacer between the first, second and/or third segments is independently 1 to 10 nucleotides in length. 
     
     
         136 . The sample, slide or kit of any one of  claims 116 - 135 , wherein the third segment of the COM optionally comprises a spacer between the plurality of label amplifier anchor segments, wherein the spacer is independently 1 to 10 nucleotides in length. 
     
     
         137 . The sample, slide or kit of any one of  claims 116 - 136 , wherein the plurality of COMs bound to the SPM and NPM is in the range of 2 to 20. 
     
     
         138 . The sample, slide or kit of any one of  claims 116 - 137 , wherein the plurality of LMs bound to the COM is in the range of 2 to 20. 
     
     
         139 . The sample, slide or kit of any one of  claims 116 - 138 , wherein the plurality of LPs bound to the LM is in the range of 2 to 20.