Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6813—Hybridisation assays
  • C12Q1/6841—In situ hybridisation
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
  • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
  • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N1/00—Sampling; Preparing specimens for investigation
  • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
  • G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis

Definitions

• the invention provides methods of detecting a double stranded nucleic acid.
• the invention provides a method of detecting a double stranded nucleic acid comprising (A) contacting a sample comprising a cell comprising one or more double stranded nucleic acids with a target probe set, wherein the target probe set comprises a pair of target probes comprising a first probe that specifically hybridizes to a first strand of a target double stranded nucleic acid, and a second probe that specifically hybridizes to the second strand of the double stranded nucleic acid; (B) contacting the sample with a pre-amplifier comprising a binding site for the first target probe, a binding site for the second target probe, and a plurality of binding sites for an amplifier; (C) contacting the sample with a plurality of amplifiers, wherein the amplifiers comprise a binding site for the pre-amplifier and a plurality of binding sites for a label probe; (D)
• the target probe set comprises two or more pairs of target probes that specifically hybridize to the same target nucleic acid.
• the double stranded nucleic acid is DNA, RNA, or a DNA/RNA hybrid.
• the sample is a tissue specimen or is derived from a tissue specimen.
• the sample is a blood sample or is derived from a blood sample.
• the sample is a cytological sample or derived from a cytological sample.
• the inventions provides a sample of fixed and permeabilized cells, comprising (A) at least one fixed and permeabilized cell containing a target double stranded nucleic acid; (B) a target probe set, wherein the target probe set comprises a pair of target probes comprising a first probe specifically hybridized to a first strand of a target double stranded nucleic acid, and a second probe specifically hybridized to the second strand of the double stranded nucleic acid; (C) a pre-amplifier comprising a binding site for the first target probe, a binding site for the second target probe, and a plurality of binding sites for an amplifier, wherein the pre-amplifier is hybridized to the first and second target probes; (D) a plurality of amplifiers, wherein the amplifiers comprise a binding site for the pre-amplifier and a plurality of binding sites for a label probe, and wherein the amplifiers are hybridized to the pre-amplifier; and (E
• the target probe set comprises two or more pairs of target probes that specifically hybridize to the same target nucleic acid.
• the double stranded nucleic acid is DNA, RNA, or a DNA/RNA hybrid.
• the sample is a tissue specimen or is derived from a tissue specimen.
• the sample is a blood sample or is derived from a blood sample.
• the sample is a cytological sample or derived from a cytological sample.
• the invention provides a 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 double stranded nucleic acid; (B) a target probe set, wherein the target probe set comprises a pair of target probes comprising a first probe specifically hybridized to a first strand of a target double stranded nucleic acid, and a second probe specifically hybridized to the second strand of the double stranded nucleic acid; (C) a pre-amplifier comprising a binding site for the first target probe, a binding site for the second target probe, and a plurality of binding sites for an amplifier, wherein the pre-amplifier is hybridized to the first and second target probes; (D) a plurality of amplifiers, wherein the amplifiers comprise a binding site for the pre-amplifier and a plurality of binding sites for a label probe, and wherein the amplifiers are
• the target probe set comprises two or more pairs of target probes that specifically hybridize to the same target nucleic acid.
• the double stranded nucleic acid is DNA
• RNA or a DNA/RNA hybrid.
• the sample is a tissue specimen or is derived from a tissue specimen.
• the sample is a blood sample or is derived from a blood sample.
• the sample is a cytological sample or derived from a cytological sample.
• the invention provides a kit for detection of a target double stranded nucleic acid, comprising (A) a pre-amplifier comprising a binding site for the first target probe, a binding site for the second target probe, and a plurality of binding sites for an amplifier; (B) a plurality of amplifiers, wherein the amplifiers comprise a binding site for the pre-amplifier and a plurality of binding sites for a label probe; and (C) a plurality of label probes, wherein the label probe comprises a label and a binding site for the amplifiers.
• the sample is a tissue specimen or is derived from a tissue specimen.
• the sample is a blood sample or is derived from a blood sample.
• the sample is a cytological sample or is derived from a cytological sample.
• the invention provides a sample of fixed and permeabilized cells, comprising (A) at least one fixed and permeabilized cell containing a target double stranded nucleic acid; (B) a target probe set, wherein the target probe set comprises a pair of target probes comprising a first probe specifically hybridized to a first strand of a target double stranded nucleic acid, and a second probe specifically hybridized to the second strand of the double stranded nucleic acid; (C) a set of pre-pre-amplifiers comprising a first and second pre-pre-amplifier, wherein the first pre-pre-amplifier comprises a binding site for the first target probe, wherein the second pre-pre-amplifier comprises a binding site for the second target probe, wherein the first and second pre-pre-amplifiers comprise a plurality of binding sites for a pre-amplifier, and wherein the pre-pre-amplifiers are hybridized to the first and second target probes; (A) at least one fixed and
• the pre-amplifier comprises a binding site for the first and second pre-pre-amplifiers, wherein the melting temperature between the binding to both pre-pre-amplifiers is higher than the melting temperature between the binding of only one pre-pre-amplifier.
• the target probe set comprises two or more pairs of target probes that specifically hybridize to the same target nucleic acid.
• the sample is a tissue specimen or is derived from a tissue specimen.
• the sample is a blood sample or is derived from a blood sample.
• the sample is a cytological sample or is derived from a cytological sample.
• the invention provides a method of detecting a double stranded nucleic acid comprising: (A) contacting a sample comprising a cell comprising one or more double stranded nucleic acids with a target probe set, wherein the target probe set comprises a pair of target probes comprising a first probe that specifically hybridizes to a first strand of a target double stranded nucleic acid, and a second probe that specifically hybridizes to the second strand of the double stranded nucleic acid; (B) contacting the sample with a pre- pre-amplifier comprising a binding site for the first target probe, a binding site for the second target probe, and a plurality of binding sites for a pre-amplifier; (C) contacting the sample with a plurality of pre-amplifiers comprising a binding site for the pre-pre-amplifier, and a plurality of binding sites for an amplifier; (D) contacting the sample with a plurality of amplifiers,
• the target probe set comprises two or more pairs of target probes that specifically hybridize to the same target nucleic acid.
• the sample is a tissue specimen or is derived from a tissue specimen.
• the sample is a blood sample or is derived from a blood sample.
• the sample is a cytological sample or is derived from a cytological sample.
• the sample is a tissue specimen or is derived from a tissue specimen.
• the sample is a blood sample or is derived from a blood sample.
• the sample is a cytological sample or is derived from a cytological sample.
• the invention provides a 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 double stranded nucleic acid; (B) a target probe set, wherein the target probe set comprises a pair of target probes comprising a first probe specifically hybridized to a first strand of a target double stranded nucleic acid, and a second probe specifically hybridized to the second strand of the double stranded nucleic acid; (C) a pre-pre-amplifier comprising a binding site for the first target probe, a binding site for the second target probe, and a plurality of binding sites for a pre-amplifier, wherein the pre-pre- amplifier is hybridized to the first and second target probes; (D) a plurality of pre-amplifiers comprising a binding site for the pre-pre-amplifier and a plurality of binding sites for an amplifier, wherein
• RNA or a DNA/RNA hybrid.
• the sample is a tissue specimen or is derived from a tissue specimen.
• the sample is a blood sample or is derived from a blood sample.
• the sample is a cytological sample or is derived from a cytological sample.
• the invention provides a kit for detection of a target double stranded nucleic acid, comprising (A) a pre-pre-amplifier comprising a binding site for the first target probe, a binding site for the second target probe, and a plurality of binding sites for a pre-amplifier; (B) a pre-amplifier comprising a binding site for the pre-pre-amplifier and a plurality of binding sites for an amplifier; (C) a plurality of amplifiers, wherein the amplifiers comprise a binding site for the pre-amplifier and a plurality of binding sites for a label probe; and (D) a plurality of label probes, wherein the label probe comprises a label and a binding site for the amplifiers.
• the kit comprises a target probe set, wherein the target probe set comprises a pair of target probes comprising a first probe that specifically hybridizes to a first strand of a target double stranded nucleic acid, and a second probe that specifically hybridizes to the second strand of the target double stranded nucleic acid.
• the target probe set comprises two or more pairs of target probes that specifically hybridize to the same target nucleic acid.
• the kit comprises at least one reagent for permeabilizing cells.
• Figure 1 shows a schematic of probe designs (double strand probes (ds-probes)) for detecting double stranded DNA, RNA or DNA/RNA hybrid. For each probe pair
• the target-binding region of one probe binds to the sense strand whereas that of the other probe binds to the anti-sense strand of double stranded DNA, RNA or DNA-RNA hybrid. Simultaneous binding of each probe of the pair forms a binding site for the pre-amplifier (Panels A and B). Panels A and B show two alternative exemplary probe orientations. Panel C shows adaption of the same probe design for the BaseScopeTM signal amplification system (Baker et al., Nat. Commun. 8(1): 1998, doi: 10.1038/s41467-017-02295- 5 (2017)).
• Panel C depicts binding of each member of the pair of target probes to the sense and antisense strands of the target nucleic acid, respectively.
• Each target probe binds to a separate pre-pre-amplifier (Panel C).
• one or more probe pairs can be designed to target the same double stranded nucleic acid.
• Figure 2 shows a flow diagram for the steps for an exemplary in situ hybridization assay (left), and a modified assay using chemical denaturation (right) to denature double stranded nucleic acids.
• the standard protocol starts with baking a slide at 60°C followed by deparaffmization to remove the paraffin wax (“Bake+Dewax”).
• the slide is then treated with Epitope Retrieval buffer (“ER2”) at 88°C to unmask the target(s), followed by protease digestion (Protease III).
• ER2 Epitope Retrieval buffer
• target probes are hybridized to the slide (“Probe”), which is followed by sequential hybridization with pre-amplifiers, amplifiers and label probes (“AMPs”) and then detection of the target with the chromogen Fast Red (“Detection”).
• Detection The modified protocol for DNA detection adds a chemical denaturation step (“Chemical DN”,
• Figure 3 shows detection of fibroblast growth factor receptor 1 (FGFR1) genomic DNA detection requires both Z1 (sense strand binding) and Z2 (antisense strand binding) probes, which ensures specificity for double stranded DNA. DNA was detected as red punctate dots inside nuclei (blue).
• FGFR1 fibroblast growth factor receptor 1
• Figure 4 shows detection of different genes in cells (HEK293) and tissues (colon cancer, ovarian cancer, normal pancreas, normal breast) by ds-probes. DNA was detected as red punctate dots inside nuclei (blue).
• Figures 5A-5C show a schematic of previously described methods of detecting a nucleic acid target using a signal generating complex (SGC).
• SGC signal generating complex
• the methods provide for highly sensitive and specific detection of double stranded nucleic acids in a cell.
• RNAscopeTM uses specially designed oligonucleotide probes, sometimes referred to as "double-Z" or ZZ probes, in combination with a branched-DNA-like signal amplification system to reliably detect RNA as small as 1 kilobase at single-molecule sensitivity under standard bright-field microscopy (Anderson et ah, J Cell. Biochem. 117(10):2201-2208 (2016); Wang et ah, ./. Mol. Diagn. 14(l):22-29 (2012)).
• Such a probe design greatly improves the specificity of signal amplification because only when both probes in each pair bind to their intended target can signal amplification occur.
• RNAscopeTM for DNA detection is hampered by unwanted RNA detection because RNAscopeTM probes cannot discriminate DNA from RNA targets.
• RNA can be eliminated by enzymatic (for example, RNase A) and chemical (for example, NaOH) methods, adding these steps can cause significant degradation of nuclear and cellular morphology and compromise DNA detection or detection of double stranded nucleic acids in an in situ detection assay.
• RNAscopeTM RNAscopeTM signal amplification system without interference from RNA. This is achieved by designing each probe pair to bind to both strands of double stranded nucleic acid, such as double stranded DNA, RNA or DNA/RNA hybrid. Single stranded RNA or DNA will not be detected because only one of the two probes in each pair will bind to it, which effectively prevents signal amplification and detection of single stranded RNA and single stranded DNA targets.
• the assay strategy allows highly sensitive and specific detection of very short DNA sequences (as short as lkb), providing a ⁇ 100-fold higher resolution than current DNA FISH assays.
• the methods of the invention selectively detect double stranded DNA, which make it possible to distinguish double stranded DNA from single stranded DNA.
• the methods of the invention are also applicable to distinguishing double stranded RNA and/or RNA-DNA hybrid from single stranded DNA and/or RNA.
• this method preserves RNA when detecting DNA, the methods can be used for simultaneous detection of RNA and DNA targets in the same sample of cells or tissue section.
• the present invention extends the probe design principle at the core of the
• RNAscopeTM technology Wang et al., supra , 2012 to double stranded nucleic acid, such as DNA, detection by requiring each probe pair to bind both strands of a double stranded nucleic acid, such as DNA, for signal amplification to occur ( Figure 1).
• each probe contains a sequence segment that binds to a specific sequence in the target.
• double stranded nucleic acid detection for example, DNA detection
• two probes bind to adjacent sites on opposite strands in the target double stranded nucleic acid.
• the“Z2” probe is similarly depicted with the target binding site 5' to the pre-pre-amplifier binding site.
• the target probe pair can independently be in either orientation, that is, one member of the pair of target probes can have the target binding site 5' or 3' to the pre-amplifier or pre-pre-amplifier binding site, and can be paired with a second probe having a binding site 5' or 3' to the pre-amplifier or pre- pre-amplifier, such that there are four possible combinations of orientations for the target probe pairs.
• the two strands are labeled as“sense” and“antisense” for the purposes of illustration. It is understood that the target probe pairs bind to opposite strands of a double stranded nucleic acid. It is further understood that the region of a double stranded nucleic acid to be bound by the target probe pair does not need to be in a coding region, for example, a coding region of DNA having sense and antisense strands, but can be in a non coding region.
• the methods of the invention disclosed herein can be used to detect any double stranded nucleic acid, such as DNA biomarkers, in various cells and tissue types.
• the ds-probe design is used with RNAscopeTM to detect multiple gene targets in cells and tissues (see Example 1 and Figure 4).
• the methods of the invention can be used to detect and characterize a variety of DNA structural variations such as deletion, insertion, gene fusion, translocation, duplication, and amplification found in neoplasms and other diseases.
• the methods of the invention can also be used to detect double stranded viral DNA and RNA and bacterial DNA with double-strand specificity.
• the target probe thus includes a first polynucleotide sequence that is complementary to a polynucleotide sequence of the target nucleic acid and a second polynucleotide sequence that is complementary to a polynucleotide sequence of the label probe, amplifier, pre-amplifier, pre-pre-amplifier, or the like.
• the target probe binds to a pre-amplifier, as in Figures 1 A and IB, or to a pre-pre-amplifier, as in Figure 1C.
• the target probe is generally single stranded so that the complementary sequence is available to hybridize with a corresponding target nucleic acid, label probe, amplifier, pre-amplifier or pre-pre-amplifier.
• the target probes are provided as a pair, wherein one member of the pair binds to one strand of the double stranded nucleic acid, and the other target probe binds to the opposite strand of the double stranded nucleic acid.
• an "amplifier” is a molecule, typically a polynucleotide, that is capable of hybridizing to multiple label probes. Typically, the amplifier hybridizes to multiple identical label probes. The amplifier can also hybridize to a target nucleic acid, to at least one target probe of a pair of target probes, to both target probes of a pair of target probes, or to nucleic acid bound to a target probe such as an amplifier, pre-amplifier or pre-pre- amplifier. For example, the amplifier can hybridize to at least one target probe and to a plurality of label probes, or to a pre-amplifier and a plurality of label probes.
• the amplifier can hybridize to a pre-amplifier.
• the amplifier can be, for example, a linear, forked, comb-like, or branched nucleic acid.
• the amplifier can include modified nucleotides and/or nonstandard internucleotide linkages as well as standard deoxyribonucleotides,
• Suitable amplifiers are described, for example, in U.S. Patent Nos. 5,635,352, 5,124,246, 5,710,264, 5,849,481, and 7,709,198 and U.S.
• the amplifier binds to a pre-amplifier and label probes (see Figure 5).
• a "pre-amplifier” is a molecule, typically a polynucleotide, that serves as an intermediate binding component between one or more target probes and one or more amplifiers. Typically, the pre-amplifier hybridizes simultaneously to one or more target probes and to a plurality of amplifiers. Exemplary pre-amplifiers are described, for example, in U.S. Patent Nos. 5,635,352, 5,681,697 and 7,709,198 and U.S. publications 2008/0038725, 2009/0081688 and 2017/0101672, each of which is incorporated by reference.
• a pre-amplifier binds to both members of a target probe pair (see Figure 1 A, IB and 5A), to a pre-pre-amplifier that can bind to a target probe pair ( Figure 5B), or to both members of a pair of pre-pre-amplifiers that can bind to a target probe pair (see Figure 5C).
• a pre-amplifier also binds to an amplifier (see Figure 5).
• a "pre-pre-amplifier” is a molecule, typically a polynucleotide, that serves as an intermediate binding component between one or more target probes and one or more pre-amplifiers. Typically, the pre-pre-amplifier hybridizes simultaneously to one or more target probes and to a plurality of pre-amplifiers. Exemplary pre-pre-amplifiers are described, for example, in 2017/0101672, which is incorporated by reference.
• a pre-pre-amplifier binds to a target probe pair (see Figure 5B) or to a member of a target probe pair (see Figures 1C and 5C) and to a pre-amplifier (see Figure 5C).
• the invention provides a method of detecting a double stranded nucleic acid comprising: (A) contacting a sample comprising a cell comprising one or more double stranded nucleic acids with a target probe set, wherein the target probe set comprises a pair of target probes comprising a first probe that specifically hybridizes to a first strand of a target double stranded nucleic acid, and a second probe that specifically hybridizes to the second strand of the double stranded nucleic acid; (B) contacting the sample with a pre-amplifier comprising a binding site for the first target probe, a binding site for the second target probe, and a plurality of binding sites for an amplifier; (C) contacting the sample with a plurality of amplifiers, wherein the amplifiers comprise a binding site for the pre-amplifier and a plurality of binding sites for a label probe; (D) contacting the sample with a plurality of label probes, wherein the label probe
• the double stranded nucleic acid is DNA, RNA, or a
• the pre-amplifier comprises a binding site for the first and second pre-pre-amplifiers, wherein the melting temperature between the binding to both pre- pre-amplifiers is higher than the melting temperature between the binding of only one pre-pre- amplifier.
• the target probe set comprises two or more pairs of target probes that specifically hybridize to the same target nucleic acid.
• the double stranded nucleic acid is DNA, RNA, or a
• the sample is a tissue specimen or is derived from a tissue specimen.
• the sample is a blood sample or is derived from a blood sample.
• the sample is a cytological sample or is derived from a cytological sample.
• the invention provides a method of detecting a double stranded nucleic acid comprising (A) contacting a sample comprising a cell comprising one or more double stranded nucleic acids with a target probe set, wherein the target probe set comprises a pair of target probes comprising a first probe that specifically hybridizes to a first strand of a target double stranded nucleic acid, and a second probe that specifically hybridizes to the second strand of the double stranded nucleic acid; (B) contacting the sample with a pre- pre-amplifier comprising a binding site for the first target probe, a binding site for the second target probe, and a plurality of binding sites for a pre-amplifier; (C) contacting the sample with a plurality of pre-amplifiers comprising a binding site for the pre-pre-amplifier, and a plurality of binding sites for an amplifier; (D) contacting the sample with a plurality of amplifiers, where
• the target probe set comprises two or more pairs of target probes that specifically hybridize to the same target nucleic acid.
• the double stranded nucleic acid is DNA, RNA, or a DNA/RNA hybrid.
• the sample is a tissue specimen or is derived from a tissue specimen.
• the sample is a blood sample or is derived from a blood sample.
• the sample is a cytological sample or is derived from a cytological sample.
• each target probe set that is specific for a target nucleic acid comprises two or more pairs of target probes that specifically hybridize to the same target double stranded nucleic acid.
• the pairs of target probes in the target probe set specific for a target nucleic acid bind to different and non-overlapping sequences of the target nucleic acid.
• the molecule that binds to the target probe pairs either a pre-amplifier (see Figures 1 A, IB and 5 A), or a pre-pre-amplifier (see Figures 1C, 5B and 5C), generally are the same for target probe pairs in the same target probe set.
• the target probe pairs that bind to the same target double stranded nucleic acid can be designed to comprise the same binding site for the molecule in the SGC that binds to the target probe pairs, that is, a pre-amplifier or pre-pre- amplifier.
• the use of multiple target probe pairs to detect a target nucleic acid provides for a higher signal associated with the assembly of multiple SGCs on the same target nucleic acid.
• the number of target probe pairs used for binding to the same target nucleic acid are in the range of 1-10, 1-20, 1-30, 1-40, 1-50, 1-60, 1-70, 1-80, 1-90, 1-100, 1- 110, 1-120, 1-130, 1-140, 1-150, 1-160, 1-170, 1-180, 1-190, or 1-200 pairs per target, or larger numbers of pairs, or any integer number of pairs in between, for example, 1, 2, 3, 4, 5,
• a target nucleic acid is detected with a plurality of target probe pairs.
• target probe pairs are designed to bind to more than one region of a target nucleic acid to allow for the assembly of multiple SGCs onto a target nucleic acid. It is understood that the target binding sites of one target probe pair do not overlap with the target binding sites of another target probe pair if a plurality of target probe pairs are being used to bind to the same target nucleic acid.
• the target nucleic acids detected by the methods of the invention can be any double stranded target nucleic acid present in the cell sample.
• the target nucleic acids can independently be DNA, double stranded RNA or DNA/RNA hybrids.
• the target nucleic acids to be detected can be, but are not necessarily, the same type of nucleic acid.
• the methods of the invention can be combined with methods for detecting single stranded nucleic acids, such as RNAscopeTM or BaseScopeTM, such that both double stranded and single stranded nucleic acids can be detected in the same sample.
• ISH, FISH and CISH methods are well known to those skilled in the art (see, for example, Stoler, Clinics in Laboratory Medicine 10(1):215-236 (1990); In situ hybridization. A practical approach , Wilkinson, ed., IRL Press, Oxford (1992); Schwarzacher and Heslop-Harrison, Practical in situ hybridization , BIOS Scientific Publishers Ltd, Oxford (2000)).
• the cell is optionally fixed and/or permeabilized before hybridization of the target probes. Fixing and
• permeabilizing cells can facilitate retaining the nucleic acid targets in the cell and permit the target probes, label probes, amplifiers, pre-amplifiers, pre-pre-amplifiers, and so forth, to enter the cell and reach the target nucleic acid molecule.
• the cell is optionally washed to remove materials not captured to a nucleic acid target.
• the cell can be washed after any of various steps, for example, after hybridization of the target probes to the nucleic acid targets to remove unbound target probes, after hybridization of the pre-pre-amplifiers, pre-amplifiers, amplifiers, and/or label probes to the target probes, and the like.
• Such samples can be, but are not limited to, organs, tissues, tissue fractions and/or cells isolated from an organism such as a mammal.
• Exemplary biological samples include, but are not limited to, a cell culture, including a primary cell culture, a cell line, a tissue, an organ, an organelle, a biological fluid, and the like.
• Additional biological samples include but are not limited to a skin sample, tissue biopsies, including fine needle aspirates, cytological samples, stool, bodily fluids, including blood and/or serum samples, saliva, semen, and the like. Such samples can be used for medical or veterinary diagnostic purposes.
• the sample is a tissue specimen or is derived from a tissue specimen.
• the sample is a blood sample or is derived from a blood sample.
• the sample is a cytological sample or is derived from a cytological sample.
• the invention is based on building a complex between a target double stranded nucleic acid in order to label the double stranded nucleic acid with a detectable label.
• a complex is sometimes referred to as a signal generating complex (SGC; see, for example, US 20170101672).
• SGC signal generating complex
• Such a complex, or SGC is achieved by building layers of molecules that allow the attachment of a large number of labels to a target double stranded nucleic acid.
• the methods of the invention can employ a signal generating complex (SGC), where the SGC comprises multiple molecules rather than a single molecule.
• SGC signal generating complex
• Such an SGC is particularly useful for amplifying the detectable signal, providing higher sensitivity detection of target nucleic acids.
• Such methods for amplifying a signal are described, for example, in U.S. Patent Nos. 5,635,352, 5,124,246, 5,710,264, 5,849,481, and 7,709,198, and U.S.
• FIG. 5A A basic Signal Generating Complex (SGC) is illustrated in Figure 5A (see also US 2009/0081688, which is incorporated herein by reference).
• SGC Signal Generating Complex
• Figure 5A A pair of target probes, depicted in Figure 5 as a pair of "Z's", hybridizes to a complementary molecule sequence, labeled "Target”.
• Figure 5 depicts the target as a single line, whereas it is understood that the target in the present invention is a double stranded nucleic acid, as illustrated in Figure 1 in more detail.
• Each target probe contains an additional sequence complementary to a pre-amplifier molecule (PA, illustrated in green), which must hybridize simultaneously to both members of the target probe pair in order to bind stably.
• the pre-amplifier molecule is made up of two domains: one domain with a region that hybridizes to each target probe, and one domain that contains a series of nucleotide sequence repeats, each complementary to a sequence on the amplifier molecule (Amp, illustrated in black). The presence of multiple repeats of this sequence allows multiple amplifier molecules to hybridize to one pre-amplifier, which increases the overall signal amplification.
• Each amplifier molecule is made up of two domains, one domain with a region that hybridizes to the pre-amplifier, and one domain that contains a series of nucleotide sequence repeats, each complementary to a sequence on the label probe (LP, illustrated in yellow), allowing multiple label probes to hybridize to each amplifier molecule, further increasing the total signal amplification.
• Each label probe contains two components. One component is made up of a nucleotide sequence
• the label probe is depicted as a line, representing the nucleic acid component, and a star, representing the signal-generating component. Together, the assembly from target probe to label probe is referred to as a Signal Generating Complex (SGC).
• SGC Signal Generating Complex
• Figure 5B illustrates a SGC enlarged by adding an amplification molecule layer, in this case a pre-pre-amplifier molecule (PPA, shown in red).
• PPA pre-pre-amplifier molecule
• the PPA binds to both target probes in one domain and multiple pre-amplifiers (PAs) in another domain.
• Figure 5C illustrates a different SGC structure that uses collaborative hybridization at the pre-amplifier level (see US 2017/0101672, which is incorporated herein by reference).
• a pair of target probes hybridize to the target molecule sequence.
• Each target probe contains an additional sequence complementary to a unique pre-pre-amplifier molecule (PPA-1, illustrated in purple; PPA-2, illustrated in red).
• PPA-1 illustrated in purple
• PPA-2 illustrated in red
• Each pre-pre-amplifier molecule is made up of two domains, one domain with a region that hybridizes to one of the target probes, and one domain that contains a series of nucleotide sequence repeats, each containing both a sequence
• each PA must hybridize to both PPA molecules simultaneously.
• Each pre amplifier molecule is made up of two domains, one domain that contains sequences complementary to both pre-pre-amplifiers to allow hybridization, and one domain that contains a series of nucleotide sequence repeats each complementary to a sequence on the amplifier molecule (AMP, illustrated in black). Multiple repeats of the amplifier
• hybridization sequence allows multiple amplifier molecules to hybridize to each pre amplifier, further increasing signal amplification.
• amplifier molecules are shown hybridizing to one pre-amplifier molecule, but it is understood that amplifiers can bind to each pre-amplifier.
• Each amplifier molecule contains a series of nucleotide sequence repeats complementary to a sequence within the label probe (LP, illustrated in yellow), allowing several label probes to hybridize to each amplifier molecule.
• Each label probe contains a signal -generating element to provide for signal detection.
• the requirement that an SGC be formed only when both members of a target probe pair are bound to the target nucleic acid is achieved by requiring that a pre-amplifier be bound to both pre-pre-amplifiers, which in turn are bound to both members of the target probe pair, respectively.
• This requirement is achieved by designing the binding sites between the pre-pre-amplifiers and the pre-amplifier such that the melting temperature (Tm) between the binding of both pre-pre-amplifiers to the pre-amplifier is higher than the melting temperature of either pre-pre-amplifier alone, and where the binding of one of the pre-pre-amplifiers to the pre-amplifier is unstable under the conditions of the assay.
• oligonucleotides or probes that specifically and selectively bind to a target nucleic acid, or other components of the SGC are well known to those skilled in the art (see Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Ed., Cold Spring Harbor Laboratory, New York (2001); Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, MD (1999)).
• the target probes are designed such that the probes specifically hybridize to a target nucleic acid.
• a desired specificity can be achieved using appropriate selection of regions of a target nucleic acid as well as appropriate lengths of a binding agent such as an oligonucleotide or probe, and such selection methods are well known to those skilled in the art.
• reagents such as oligonucleotides or probes
• target nucleic acid over another target nucleic acid or over non-target nucleic acids, or to provide binding to the components of the SGC.
• “specifically hybridize,”“specifically label,” and“specifically bind” refer to hybridization, labeling or binding to a target nucleic acid but not to non-target nucleic acids, for example, another target nucleic acid or nucleic acids that are not desired to be targeted.
• embodiments of the invention include the use of target probe pairs, with each member of the pair binding to opposite strands of a double stranded nucleic acid.
• a pair of target probes binds to the same pre-amplifier ( Figures 1 A,
• a probe configuration sometimes referred to as a“Z” configuration, can be used.
• a“Z” configuration Such a configuration and its advantages for increasing sensitivity and decreasing background are described, for example, in U.S. Patent No.
• the SGC used to detect the presence of a target double stranded nucleic acid is based on a collaborative hybridization of one or more components of the SGC (see US 20170101672 and WO 2017/066211, each of which is incorporated herein by reference).
• a collaborative hybridization is also referred to herein as BaseScopeTM
• a collaborative hybridization effect the binding between two components of an SGC is mediated by two binding sites, and the melting temperature of the binding to the two sites simultaneously is higher than the melting temperature of the binding of one site alone (see US 20170101672 and WO 2017/066211).
• the collaborative hybridization effect can be enhanced by target probe set configurations as described in US 20170101672 and WO 2017/066211.
• the methods of the invention, and related compositions can utilize collaborative hybridization to increase specificity and to reduce background in in situ detection of double stranded nucleic acid targets, where a complex physiochemical environment and the presence of an overwhelming number of non-target molecules can generate high noise.
• a collaborative hybridization method the binding of label probes only occurs when the SGC is bound to the target nucleic acid.
• the method can be readily modified to provide a desired signal to noise ratio by increasing the number of collaborative hybridizations in one or more components of the SGC.
• the collaborative hybridization can be applied to various components of the SGC.
• the binding between components of an SGC can be a stable reaction, as described herein, or the binding can be configured to require a collaborative hybridization, also as described herein.
• the binding component intended for collaborative hybridization are designed such that the component contains two segments that bind to another component.
• the methods for detecting a target nucleic acid can utilize collaborative hybridization for the binding reactions between any one or all of the components in the detection system that provides an SGC specifically bound to a target nucleic acid.
• the components are generally bound directly to each other.
• the binding reaction is generally by hybridization.
• the binding between the components is direct.
• an intermediary component can be included such that the binding of one component to another is indirect , for example, the intermediary component contains complementary binding sites to bridge two other components.
• the configuration of various components can be selected to provide a desired stable or collaborative hybridization binding reaction (see, for example, US 20170101672). It is understood that, even if a binding reaction is exemplified herein as a stable or unstable reaction, such as for a collaborative hybridization, any of the binding reactions can be modified, as desired, so long as the target double stranded nucleic acid is detected. It is further understood that the configuration can be varied and selected depending on the assay and hybridization conditions to be used.
• the segments of complementary nucleic acid sequence between the components is generally in the range of 10 to 50 nucleotides, or greater, for example, 16 to 30 nucleotides, such as 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides, or greater.
• nucleic acid segments that are complementary to other nucleic acid segments can be reduced further, if desired.
• a person skilled in the art can readily determine appropriate probe designs, including length, the presence of modified nucleotides, and the like, to achieve a desired interaction between nucleic acid components.
• the complementary sequences can optionally be designed to maximize the difference in melting temperature (dT m ). This can be done by using melting temperature calculation algorithms known in the art (see, for example, SantaLucia, Proc. Natl. Acad. Sci. U.S.A. 95: 1460-1465 (1998)).
• artificial modified bases such as Locked Nucleic Acid (LNA) or bridged nucleic acid (BNA) and naturally occurring 2'-0- methyl RNA are known to enhance the binding strength between complementary pairs (Petersen and Wengel, Trends Biotechnol. 21 :74-81 (2003); Majlessi et ah, Nucl. Acids Res. 26:2224-2229 (1998)).
• LNA Locked Nucleic Acid
• BNA bridged nucleic acid
• 2'-0- methyl RNA naturally occurring 2'-0- methyl RNA
• One approach is to utilize modified nucleotides (LNA, BNA or 2'-0-methyl RNA). Because each modified base can increase the melting temperature, the length of binding regions between two nucleic acid sequences (i.e., complementary sequences) can be substantially shortened. The binding strength of a modified base to its complement is stronger, and the difference in melting temperatures (dT m ) is increased. Yet another embodiment is to use three modified bases (for example, three LNA, BNA or 2'-0-methyl RNA bases, or a combination of two or three different modified bases) in the complementary sequences of a nucleic acid component or between two nucleic acid components, for example of a signal generating complex (SGC), that are to be hybridized. Such components can be, for example, pre-pre-amplifier, a pre-amplifier, an amplifier, a label probe, or a pair of target probes.
• SGC signal generating complex
• the modified bases such as LNA or BNA
• LNA or BNA can be used in the segments of selected components of SGC, in particular those mediating binding between nucleic acid components, which increases the binding strength of the base to its complementary base, allowing a reduction in the length of the complementary segments (see, for example, Petersen and Wengel, Trends Biotechnol. 21 :74-81 (2003); US Patent No. 7,399,845).
• Artificial bases that expand the natural 4-letter alphabet such as the Artificially Expanded Genetic Information System (AEGIS; Yang et al., Nucl. Acids Res. 34 (21): 6095-6101 (2006)) can be used.
• AEGIS Artificially Expanded Genetic Information System
• the target probe pair can be designed to bind to immediately adjacent segments of the target nucleic acid or on segments that have one to a number of bases between the target probe binding sites of the target probe pair.
• target probe pairs are designed for binding to the target nucleic acid such that there are generally between 0 to 500 bases between the binding sites on the target nucleic acid, for example, 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, or 500 bases, or any integer length in between.
• the binding sites for the pair of target probes are between 0 and 100, for example, 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, or any integer length in between.
• the members of the target probe pair bind to opposite strands of the double stranded nucleic acid (see, for example, Figure 1).
• the binding sites of the pair of target probes are on opposite strands.
• the target binding sites for the pair of probes can overlap since the binding sites occur on opposite strands for the respective members of the target probe pair. It is understood that overlap can occur so long as there is no steric hindrance for the simultaneous binding of both target probes to the respective target nucleic acid strands. A person skilled in the art can readily determine permissible overlap between target binding sites on opposite strands, if such overlap is desired.
• any of a number of enzymes or non-enzyme labels can be utilized so long as the enzymatic activity or non-enzyme label, respectively, can be detected.
• the enzyme thereby produces a detectable signal, which can be utilized to detect a target nucleic acid.
• Particularly useful detectable signals are chromogenic or fluorogenic signals.
• particularly useful enzymes for use as a label include those for which a chromogenic or fluorogenic substrate is available. Such chromogenic or fluorogenic substrates can be converted by enzymatic reaction to a readily detectable chromogenic or fluorescent product, which can be readily detected and/or quantified using microscopy or spectroscopy.
• Such enzymes are well known to those skilled in the art, including but not limited to, horseradish peroxidase, alkaline phosphatase, b-galactosidase, glucose oxidase, and the like (see Hermanson, Bioconjugate Techniques , Academic Press, San Diego (1996)).
• Other enzymes that have well known chromogenic or fluorogenic substrates include various peptidases, where chromogenic or fluorogenic peptide substrates can be utilized to detect proteolytic cleavage reactions.
• chromogenic and fluorogenic substrates are also well known in bacterial diagnostics, including but not limited to the use of a- and b-galactosidase, b-glucuronidase, 6-phospho ⁇ - D-galatoside 6-phosphogalactohydrolase, b-gluosidase, a-glucosidase, amylase,
• fluorogenic substrates include, but are not limited to, 4-(Trifluoromethyl)umbelliferyl phosphate for alkaline phosphatase; 4-Methylumbelliferyl phosphate bis (2-amino- 2-methyl- 1,3 -propanediol), 4-Methylumbelliferyl phosphate bis (cyclohexylammonium) and 4- Methylumbelliferyl phosphate for phosphatases; QuantaBluTM and QuantaRedTM for horseradish peroxidase; 4-Methylumbelliferyl b-D-galactopyranoside, Fluorescein di ⁇ -D- galactopyranoside) and Naphthofluorescein di- ⁇ -D-galactopyranoside) for b-galactosidase; 3-Acetylumbelliferyl b-D-glucopyranoside and 4-Methyl umbel 1 iferyl -b- D-glucopyranoside for b
• Exemplary rare earth metals and metal isotopes suitable as a detectable label include, but are not limited to, lanthanide (III) isotopes such as 141Pr, 142Nd, 143Nd, 144Nd, 145Nd, 146Nd, 147Sm, 148Nd, 149Sm, 150Nd, 151Eu, 152Sm, 153Eu, 154Sm, 155Gd, 156Gd, 158Gd, 159Tb, 160Gd, 161Dy, 162Dy, 163Dy, 164Dy, 165Ho, 166Er, 167Er, 168Er, 169Tm, 170Er, 171Yb, 172Yb, 173Yb, 174Yb, 175Lu, and 176Yb.
• III lanthanide
• Embodiments described herein can utilize enzymes to generate a detectable signal using appropriate chromogenic or fluorogenic substrates. It is understood that, alternatively, a label probe can have a detectable label directly coupled to the nucleic acid portion of the label probe. Exemplary detectable labels are well known to those skilled in the art, including but not limited to chromogenic or fluorescent labels (see Hermanson, Bioconjugate Techniques , Academic Press, San Diego (1996)).
• the methods of the invention can utilize concurrent detection of multiple target nucleic acids.
• fluorophores as labels
• the fluorophores to be used for detection of multiple target nucleic acids are selected so that each of the fluorophores are distinguishable and can be detected concurrently in the fluorescence microscope in the case of concurrent detection of target nucleic acids.
• fluorophores are selected to have spectral separation of the emissions so that distinct labeling of the target nucleic acids can be detected concurrently.
• cytometry e.g., mass cytometry, cytometry by time of flight (CyTOF), flow cytometry
• spectroscopy can be utilized to visualize chromogenic, fluorescent, or metal detectable signals associated with the respective target nucleic acids.
• cytometry e.g., mass cytometry, cytometry by time of flight (CyTOF), flow cytometry
• spectroscopy can be utilized to visualize chromogenic, fluorescent, or metal detectable signals associated with the respective target nucleic acids.
• chromogenic substrates or fluorogenic substrates, or chromogenic or fluorescent labels, or rare earth metal isotopes will be utilized for a particular assay, if different labels are used in the same assay, so that a single type of instrument can be used for detection of nucleic acid targets in the same sample.
• the methods of the invention can be applied to multiplex detection of target double stranded nucleic acids.
• the methods of the invention are applied to the detection of two or more target nucleic acids, for example, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more target nucleic acids.
• the number of target nucleic acids that can be detected depends on the detection label. For fluorescent labels, up to 10 nucleic targets can generally be detected. For metal tagged probes using mass spectrometry-based detection, the number of target nucleic acids can be up to 150.
• a person skilled in the art can readily select suitable distinct labels to allow detection of more than one target double stranded nucleic acid in a sample.
• the invention described herein generally relates to detection of double stranded nucleic acids in a sample. It is understood that the methods of the invention can additionally be applied to detecting target nucleic acids and optionally other molecules in the sample, in particular in the same cell as the target nucleic acid.
• proteins expressed in a cell can also concurrently be detected. Detection of proteins in a cell are well known to those skilled in the art, for example, by detecting the binding of protein-specific antibodies using any of the well known detection systems, including those described herein for detection of target nucleic acids. Detection of target nucleic acids and protein have been described (see, for example, Schulz et ah, Cell Syst. 6(l):25-36 (2016)).
• the invention can be carried out in any desired order, so long as the double stranded target nucleic acid is detected.
• the steps of contacting a cell with any components for assembly of an SGC can be performed in any desired order, can be carried out sequentially, or can be carried out simultaneously, or some steps can be performed sequentially while others are performed simultaneously, as desired, so long as the target double stranded nucleic acid is detected.
• embodiments disclosed herein can be independently combined with other embodiments disclosed herein, as desired, in order to utilize various configurations, component sizes, assay conditions, assay sensitivity, and the like.
• the number of assay steps it can be desirable to reduce the number of assay steps, for example, reduce the number of hybridization and wash steps.
• One way of reducing the number of assay steps is to pre-assemble some or all components of the SGC prior to contacting with a cell. Such a pre-assembly can be performed by hybridizing some or all of the components of the SGC together prior to contacting the target nucleic acid.
• the invention also provides in one embodiment a sample of fixed and
• permeabilized cells comprising (A) at least one fixed and permeabilized cell containing a target double stranded nucleic acid; (B) a target probe set, wherein the target probe set comprises a pair of target probes comprising a first probe specifically hybridized to a first strand of a target double stranded nucleic acid, and a second probe specifically hybridized to the second strand of the double stranded nucleic acid; (C) a pre-amplifier comprising a binding site for the first target probe, a binding site for the second target probe, and a plurality of binding sites for an amplifier, wherein the pre-amplifier is hybridized to the first and second target probes; (D) a plurality of amplifiers, wherein the amplifiers comprise a binding site for the pre-amplifier and a plurality of binding sites for a label probe, and wherein the amplifiers are hybridized to the pre-amplifier; and (E) a plurality of label probes, wherein the label probe comprises a
• the double stranded nucleic acid is DNA, RNA, or a DNA/RNA hybrid.
• the invention additionally provides a slide comprising a cell or a plurality of cells.
• the cell or cells are fixed to the slide.
• the cell or cells are
• RNA or a DNA/RNA hybrid.
• the sample is a tissue specimen or is derived from a tissue specimen.
• the sample is a blood sample or is derived from a blood sample.
• the sample is a cytological sample or is derived from a cytological sample.
• the double stranded nucleic acid is DNA
• the sample is a tissue specimen or is derived from a tissue specimen.
• the sample is a blood sample or is derived from a blood sample.
• the sample is a cytological sample or is derived from a cytological sample.
• the invention provides a 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 double stranded nucleic acid; (B) a target probe set, wherein the target probe set comprises a pair of target probes comprising a first probe specifically hybridized to a first strand of a target double stranded nucleic acid, and a second probe specifically hybridized to the second strand of the double stranded nucleic acid; (C) a pre-pre-amplifier comprising a binding site for the first target probe, a binding site for the second target probe, and a plurality of binding sites for a pre-amplifier, wherein the pre-pre- amplifier is hybridized to the first and second target probes; (D) a plurality of pre-amplifiers comprising a binding site for the pre-pre-amplifier and a plurality of binding sites for an amplifier, wherein
• the target probe set comprises two or more pairs of target probes that specifically hybridize to the same target nucleic acid.
• the double stranded nucleic acid is DNA
• RNA or a DNA/RNA hybrid.
• the sample is a tissue specimen or is derived from a tissue specimen.
• the sample is a blood sample or is derived from a blood sample.
• the sample is a cytological sample or is derived from a cytological sample.
• the invention also provides a kit comprising the components of an SGC, as described herein, where the kit does not include the target nucleic acid.
• a kit can comprise pre-amplifiers (PAs), amplifiers (AMPs) and label probes (LPs), and optionally pre- pre-amplifiers (PPAs), as disclosed herein.
• the kit can comprise target probes (TPs) directed to a particular target double stranded nucleic acid, or a plurality of target double stranded nucleic acids.
• the components of a kit of the invention can optionally be in a container, and optionally instructions for using the kit can be provided.
• the invention provides a kit for detection of a target double stranded nucleic acid, comprising (A) a pre-amplifier comprising a binding site for the first target probe, a binding site for the second target probe, and a plurality of binding sites for an amplifier; (B) a plurality of amplifiers, wherein the amplifiers comprise a binding site for the pre-amplifier and a plurality of binding sites for a label probe; and (C) a plurality of label probes, wherein the label probe comprises a label and a binding site for the amplifiers.
• the kit comprises a target probe set, wherein the target probe set comprises a pair of target probes comprising a first probe that specifically hybridizes to a first strand of a target double stranded nucleic acid, and a second probe that specifically hybridizes to the second strand of the double stranded nucleic acid.
• the target probe set comprises two or more pairs of target probes that specifically hybridize to the same target nucleic acid.
• the kit comprises at least one reagent for permeabilizing cells.
• the invention provides a kit for detection of a target double stranded nucleic acid, comprising (A) a set of pre-pre-amplifiers comprising a first and second pre-pre-amplifier, wherein the first pre-pre-amplifier comprises a binding site for the first target probe, wherein the second pre-pre-amplifier comprises a binding site for the second target probe, and wherein the first and second pre-pre-amplifiers comprise a plurality of binding sites for a pre-amplifier; (B) a plurality of pre-amplifiers, wherein the pre-amplifiers comprise a binding site for the first and second pre-pre-amplifiers and a plurality of binding sites for an amplifier; (C) a plurality of amplifiers, wherein the amplifiers comprise a binding site for the pre-amplifiers and a plurality of binding sites for a label probe; and (D) a plurality of label probes, wherein the label probe comprises a label and a binding
• the pre-amplifier comprises a binding site for the first and second pre-pre-amplifiers, wherein the melting temperature between the binding to both pre-pre-amplifiers is higher than the melting temperature between the binding of only one pre-pre-amplifier.
• the kit comprises at least one reagent for permeabilizing cells.
• the invention provides kit for detection of a target double stranded nucleic acid, comprising (A) a pre-pre-amplifier comprising a binding site for the first target probe, a binding site for the second target probe, and a plurality of binding sites for a pre-amplifier; (B) a pre-amplifier comprising a binding site for the pre-pre-amplifier and a plurality of binding sites for an amplifier; (C) a plurality of amplifiers, wherein the amplifiers comprise a binding site for the pre-amplifier and a plurality of binding sites for a label probe; and (D) a plurality of label probes, wherein the label probe comprises a label and a binding site for the amplifiers.
• the kit comprises a target probe set, wherein the target probe set comprises a pair of target probes comprising a first probe that specifically hybridizes to a first strand of a target double stranded nucleic acid, and a second probe that specifically hybridizes to the second strand of the target double stranded nucleic acid.
• the target probe set comprises a pair of target probes comprising a first probe that specifically hybridizes to a first strand of a target double stranded nucleic acid, and a second probe that specifically hybridizes to the second strand of the target double stranded nucleic acid.
• the kit comprises at least one reagent for permeabilizing cells.
• This example describes in situ detection of double stranded nucleic acids.
• Tissue samples were also tested for the ability to detect genes in situ.
• Tissue samples of colon cancer, normal pancreas and normal breast were contacted with sense and antisense probes for detection of epidermal growth factor receptor (EGFR) gene and detected by in situ hybridization essentially as described above.
• EGFR epidermal growth factor receptor
• a tissue sample of ovarian cancer was contacted with sense and antisense probes for detection of E3 ubiquitin-protein ligase Mdm2 (MDM2) gene.
• MDM2 E3 ubiquitin-protein ligase Mdm2

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