US20050266459A1 - Nucleic acid probes and nucleic acid analog probes - Google Patents

Nucleic acid probes and nucleic acid analog probes Download PDF

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US20050266459A1
US20050266459A1 US11/121,086 US12108605A US2005266459A1 US 20050266459 A1 US20050266459 A1 US 20050266459A1 US 12108605 A US12108605 A US 12108605A US 2005266459 A1 US2005266459 A1 US 2005266459A1
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nucleic acid
probes
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chromosomes
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Tim Poulsen
Kirsten Nielsen
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Dako Denmark ApS
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DakoCytomation Denmark AS
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • 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
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y10/00—Nanotechnology for information processing, storage or transmission, e.g. quantum computing or single electron logic
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present invention relates to probes for use in various applications.
  • these probes may be used to detect, quantify, identify, or analyze nucleic acid molecules or other molecules that bind to the probes.
  • the invention also encompasses compositions and kits containing the probes and methods of using the probes.
  • Probe-based assays are useful in the detection, quantitation and analysis of nucleic acids by hybridizing a probe to a nucleic acid sample. Probes have long been used to analyze samples for the presence of nucleic acid from a bacteria, fungi, virus or other organism (See for example; U.S. Pat. Nos. 4,851,330, 5,288,611, 5,567,587, 5,601,984 and 5,612,183). Probe-based assays are also useful in examining genetically-based clinical conditions of interest, as discussed below.
  • FISH Fluorescence in-situ hybridization
  • the technique generally entails preparing a cytological sample, labeling probes, denaturing target chromosomes and the probe, hybridizing the probe to the target sequence, and detecting a signal.
  • the hybridization reaction fluorescently stains the target sequences so that their location, size, or number can be determined using fluorescence microscopy, flow cytometry or other suitable instrumentation.
  • chromosomes are arranged in matching pairs. Each chromosome in the pair is a sister chromosome. DNA sequences ranging from whole genomes down to several kilobases can be studied using current hybridization techniques in combination with commercially available instrumentation.
  • FISH-based staining is sufficiently distinct such that the hybridization signals can be seen both in metaphase spreads and in interphase nuclei.
  • Single and multicolor FISH, using probes have been applied to different clinical applications generally known as molecular cytogenetics, including prenatal diagnosis, leukemia diagnosis, and tumor cytogenetics.
  • the FISH technique has several advantages including ease of use, rapid results, reduced background in comparison with the radioactive labels that preceded fluorescent labels, and high sensitivity.
  • the FISH technique has several applications in the clinical setting. These applications include, for example, detection of chromosomal aneuploidy in prenatal diagnoses, hematological cancers, and solid tumors; detection of gene abnormalities such as oncogene amplifications, gene deletions, or gene fusions; chromosomal structural abnormalities such as translocations, duplications, insertions, or inversions; detection of contiguous gene syndromes such as microdeletion syndrome; monitoring the genetic effect of therapy; detection of viral nucleic acids in somatic cells and viral integration sites in chromosomes; gene mapping; and cell cycle analysis (Luke and Shepelsky, Cell Vision 5:49-53 (1998)).
  • FISH FISH-based karyotyping chromosomes
  • FISH multiplex FISH
  • m-FISH Speicher et al., Nat. Genet., 12:368-75
  • SKY spectral karyotyping
  • COBRA binary ration labeling
  • color-changing karyotyping Henegariu et al., Nat. Genet., 23:263-64 (1999)
  • FISH-based banding technologies such as cross-species color banding (Müller et al., Hum.
  • SKY uses charge coupled device (CCD) imaging and Fourier spectroscopy to assess the spectrum of fluorescent wavelengths for each pixel scanned, assigning a specific pseudocolor depending on the spectrum identified (Schrock et al. Science, 273:494-497 (1996)). See also the E.C.A. Newsletter for a general description of SKY, available at http://www.biologia.uniba.it/eca/NEWSLETTERS/NS-7/02-Multiplex-FISH.html.
  • CCD charge coupled device
  • the fused signal will remain on the normal sister chromosome.
  • one probe migrates to a completely different location, where the individual signal of that translocated probe becomes apparent.
  • the other individual probe remains on the split chromosome and, because it is no longer proximal to the other probe, emits its individual signal as well.
  • the two probes are no longer juxtaposed, allowing the fused signal they cause together to split into the individual signals for each probe. See generally WO 98/51817.
  • Fusion-signal FISH is similar to ssFISH in that two probes with two different, distinguishable labels are used such that proximity of the two labels produces a new fused signal.
  • the two methods differ in that for fusion-signal FISH, the two probes bind to two different locations that are suspected to become proximal to each other as a result of a chromosomal translocation.
  • a normal sample only signals from the individual probes are present and no fused signal appears.
  • the normal chromosomes in each of the two pairs involved will still emit each of the individual probe signals.
  • fusion-signal FISH In the abnormal chromosome, in which the probes are now proximal due to the translocation, the fused signal appears.
  • FISH in situ hybridization
  • CISH chromogenic in situ hybridization
  • the invention provides a series of probes that may be used in the above-described assays or in other probe-based assays.
  • probe-based assays See Veltman et al., Biotechniques, 35:1066-70 (2003).
  • the probe flanks the breakpoint region of a chromosome. In another embodiment, the probe partially overlaps the breakpoint region of a chromosome. In another embodiment, the probe extends past the breakpoint region of a chromosome.
  • unlabeled probes are used to remove complementary labeled probes that are at a chromosomal breakpoint, thus producing labeled break point probes.
  • two differentially labeled probes extending into a chromosome breakpoint region may overlap each other.
  • the differentially labeled probes may mask the presence of the translocation due to this overlap in probe sequence.
  • the overlapping probes may instead give a signal that combines the two labels.
  • a blocking probe may be prepared from various vectors including, but not limited to, a plasmid, a cosmid, a PAC, a BAC, or a YAC.
  • a blocking probe may be unlabeled or may comprise a detectable labeled.
  • the nucleic acid probe is selected from (a) a nucleic acid comprising the nucleotide sequence substantially as set out in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ. ID NO.
  • CMML chronic myelomonocytic leukemia
  • AML acute myeloid leukemia
  • ALL acute lymphoblastic leukemia
  • ETV6 and AML1 also know as RUNX1 and CBFA2
  • RUNX1 and CBFA2 also know as RUNX1 and CBFA2
  • the ETV6/AML 1 translocation involves the ETV6 disrupting the gene between the HLH motif and the ETS DNA-binding domain.
  • CMML chronic myelomonocytic leukemia
  • MDS chronic myelomonocytic leukemia
  • ALL chronic myeloblastic leukemia
  • AML acute myelocytic leukemia
  • CML chronic myelogenous leukemia
  • T-LGL large granular lymphocyte leukemia
  • HES Hypereosinophilic syndrome
  • CMML chronic myelomonocytic leukemia
  • MDS myelodysplastic syndrome
  • ALL acute lymphoblastic leukemia
  • AML acute myelocytic leukemia
  • CML chronic myelogenous leukemia
  • T-LGL T-cell prolymphocytic leukemia
  • HES Hypereosinophilic syndrome
  • kits or compositions containing the probes of the invention wherein the kit or composition may be used to diagnose the aforementioned diseases or any other disease also associated with the target sequences bound by the claimed probes.
  • FIG. 1 shows using an unlabeled blocking probe to block the binding of labeled probes to a chromosome breakpoint.
  • two “red probes” are depicted at the top left using thin lines
  • four “green probes” are depicted at the top right using thick lines.
  • the “blocking probe” is depicted using medium-weight lines.
  • lighter shaded areas represent the signal from the first label (for example, red)
  • darker shaded areas represent the signal from the second label (for example, green)
  • unshaded areas represent co-localized (for example, green/red) signals.
  • FIG. 2 shows hybridization of BCR probes to metaphase chromosomes.
  • A Drawing of the area covered by the probes, with different colored probes denoted by short and long dashed lines;
  • B Hybridization of the BCR probes to metaphase spreads from normal peripheral blood.
  • Two normal BCR loci on chromosomes 22 are represented by two light colored dots (e.g. yellow, from the superimposition of green and red signals) or co-localized different colored signals (e.g. green and red);
  • C Hybridization of the BCR probes to nuclei from normal peripheral blood.
  • Two normal BCR loci are represented by two light colored dots or co-localized different colored signals;
  • D Hybridization of the BCR probes to metaphase spreads from the cell line BV173.
  • One normal BCR locus on chromosome 22 is represented by a light colored dot or co-locolized signals from two differently colored probes (see the bright dots at the top left of the figure).
  • Two derivative chromosomes resulting from a translocation between chromosome 9 and 22 (in cell line BV173) can be detected.
  • An additional chromosome 9 can be detected as a result of an amplification of the derivative chromosome 9;
  • E Hybridization of the BCR probes to nuclei from the cell line BV173.
  • One normal BCR locus is represented by a light colored dot or co-locolized signals from two different colored probes. (See the brightly colored dots at the top right side of the nuclei pictured here.)
  • Two derivative chromosomes resulting from a translocation between chromosome 9 and 22 can be detected.
  • An additional chromosome 9 can be detected as a result of an amplification of the derivative chromosome 9.
  • FIG. 3 shows hybridization of ETV6 probes to metaphase chromosomes.
  • A Drawing of the area cover by the probes, with different colored probes denoted by short and long dashed lines;
  • B Hybridization of the ETV6 probes to metaphase spreads from normal peripheral blood.
  • Two normal ETV6 loci on chromosomes 12 are represented by two light colored dots or co-locolized different color signals;
  • C Hybridization of the ETV6 probes to nuclei from normal peripheral blood. Two normal ETV6 loci are represented by two light colored dots or co-locolized different colored signals;
  • D Hybridization of the ETV6 probes to metaphase spreads from the cell line REH.
  • the normal ETV6 locus on chromosome 12 is deleted, represented by the lack of a light colored dot or co-locolized different colored signals. Instead, each signal shown on this spread is one pure color (green or red). Two derivative chromosomes resulting from a translocation between chromosome 12 and 21 (in cell line REH) can be detected. A small green signal can be detected as a remaining part of the deleted ETV6 locus; (E) Hybridization of the ETV6 probes to nuclei from the cell line REH.
  • the normal ETV6 locus on chromosome 12 is deleted, represented by the lack of a light colored dot or co-locolized different color signals.
  • Two derivative chromosomes resulting from a translocation between chromosome 12 and 21 can be detected.
  • a small green signal can be detected as a remaining part of the deleted ETV6 locus (top right and bottom left dots on this spread).
  • FIG. 4 shows hybridization of MLL probes to metaphase chromosomes.
  • A Drawing of the area cover by the probes, with different colored probes denoted by short and long dashed lines;
  • B Hybridization of the MLL probes to metaphase spreads from normal peripheral blood.
  • Two normal MLL loci on chromosomes 11 are represented by two light colored dots or co-locolized different colored signals (all four dots shown);
  • C Hybridization of the MLL probes to nuclei from normal peripheral blood. Two normal MLL loci are represented by two light colored dots or co-locolized different colored signals (all dots shown);
  • D Hybridization of the MLL probes to metaphase spreads from the cell line RS-4;11.
  • One normal MLL locus on chromosome 11 is represented by a light colored dot or co-locolized different colored signals (at top right). Two derivative chromosomes resulting from a translocation between chromosome 4 and 11 (in cell line RS-4;11) can be detected; (E) Hybridization of the MLL probes to nuclei from the cell line RS-4;11.
  • One normal MLL locus on chromosome 11 is represented by a light colored dot or co-locolized different colored signals (larger dots on the spread at top right and at bottom). Two derivative chromosomes resulting from a translocation between chromosome 4 and 11 (in cell line RS-4; 11) can be detected.
  • One normal TCF3 locus on chromosome 19 is represented by a light colored dot or co-locolized different colored signals (two bright dots at right). Two derivative chromosomes resulting from a translocation between chromosome 1 and 19 (in cell line 697) can be detected; (E) Hybridization of the TCF3 probes to nuclei from the cell line 697.
  • One normal TCF3 locus on chromosome 19 is represented by a light colored dot or co-locolized different colored signals (central juxtaposed dots on the spread). Two derivative chromosomes resulting from a translocation between chromosome 1 and 19 (in cell line 697) can be detected.
  • FIG. 6 shows hybridization of TLX3 probes to metaphase chromosomes.
  • A Drawing of the area cover by the probes, with different colored probes denoted by short and long dashed lines;
  • B Hybridization of the TLX3 probes to metaphase spreads from normal peripheral blood.
  • Two normal TLX3 loci on chromosomes 5 are represented by two light colored dots or co-locolized different colored signals (all dots shown here);
  • C Hybridization of the TLX3 probes to nuclei from normal peripheral blood.
  • Two normal TLX3 loci are represented by two light colored dots or co-locolized different colored signals;
  • D Hybridization of the TLX3 probes to metaphase spreads from the cell line HPB-ALL.
  • One normal TLX3 locus on chromosome 5 is represented by a light colored dot or co-locolized different colored signals (at top right).
  • Two derivative chromosomes resulting from a translocation between chromosome 5 and 14 can be detected;
  • E Hybridization of the TLX3 probes to nuclei from the cell line HPB-ALL.
  • TLX3 locus on chromosome 5 is represented by a light colored dot or co-locolized different colored signals (dot at center).
  • Two derivative chromosomes resulting from a translocation between chromosome 5 and 14 can be detected.
  • FIGS. 1-6 Color versions of FIGS. 1-6 were submitted with U.S. Provisional Application No. 60/567,570, which is incorporated herein by reference and to which this application claims priority.
  • nucleic acid refers to a nucleobase sequence-containing oligomer, polymer, or polymer segment, having a backbone formed solely from naturally occurring nucleotides or unmodified nucleotides.
  • nucleic acid analog means an oligomer, polymer, or polymer segment composed of at least one modified nucleotide, or subunits derived directly from a modification of nucleotides.
  • Non-limiting examples of naturally occurring nucleobases include: adenine, cytosine, guanine, thymine, and uracil.
  • Non-limiting examples of modified nucleotides include: 5-propynyl-uracil, 2-thio-5-propynyl-uracil, 5-methylcytosine, pseudoisocytosine, 2-thiouracil and 2-thiothymine, 2-aminopurine, N9-(2-amino-6-chloropurine), N9-(2,6-diaminopurine), hypoxanthine, N9-(7-deaza-guanine), N9-(7-deaza-8-aza-guanine) and N8-(7-deaza-8-aza-adenine).
  • Other non-limiting examples of suitable nucleobases include those nucleobases illustrated in FIGS. 2 (A) and 2 (B) of Buchardt et al. (U.S. Pat. No. 6,357,
  • nucleic acid analog also refers to synthetic molecules that can bind to a nucleic acid.
  • a nucleic acid analog probe may be comprised of peptide nucleic acids (PNAs), locked nucleic acids (LNAs), or any derivatized form of a nucleic acid.
  • PNAs peptide nucleic acids
  • LNAs locked nucleic acids
  • peptide nucleic acid or “PNA” means any oligomer or polymer comprising at least one or more PNA subunits (residues), including, but not limited to, any of the oligomer or polymer segments referred to or claimed as peptide nucleic acids in U.S. Pat. Nos.
  • PNA also applies to any oligomer or polymer segment comprising two or more subunits of those nucleic acid mimics described in the following publications: Lagriffoul et al., Bioorganic & Medicinal Chemistry Letters, 4:1081-1082 (1994); Petersen et al., Bioorganic & Medicinal Chemistry Letters, 6: 793-796 (1996); Diderichsen et al., Tett. Lett. 37: 475-478 (1996); Fujii et al., Bioorg. Med. Chem. Lett. 7: 637-627 (1997); Jordan et al., Bioorg. Med. Chem. Lett. 7: 687-690 (1997); Krotz et al., Tett.
  • LNA locked nucleic acid
  • LNA subunit means a ribonucleotide containing a methylene bridge that connects the 2′-oxygen of the ribose with the 4′-carbon. See generally, Kurreck, Eur. J. Biochem., 270:1628-44 (2003).
  • nucleic acid means that a relevant sequence is 100% identical to the non-repetitive region of a given sequence.
  • substantially identical means that a relevant sequence is at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to the non-repetitive region of a given sequence.
  • sequences may be allelic variants, sequences derived from various species, or they may be derived from the given sequence by truncation, deletion, amino acid substitution or addition.
  • the length of comparison sequences will generally be at least 50, 100, 150, 300, or more nucleotides.
  • Percent identity between two sequences is determined by standard alignment algorithms such as, for example, Basic Local Alignment Tool (BLAST) described in Altschul et al., J. Mol. Biol., 215:403-410 (1990), the algorithm of Needleman et al., J. Mol. Biol., 48:444-453 (1970), or the algorithm of Meyers et al., Comput. Appl. Biosci., 4:11-17 (1988).
  • BLAST Basic Local Alignment Tool
  • the nucleic acid probe is selected from (a) a nucleic acid comprising at least one nucleotide sequence substantially as set out in Table 2; and (b) a nucleic acid that hybridizes to the complement of the nucleic acid of (a) under low stringency conditions.
  • nucleic acid probes of the invention are identical to at least one of the sequences identified in Table 2.
  • a probe binds to a target sequence under certain conditions.
  • the term “bind” is synonymous with “hybridize.” When two molecules hybridize, they form a combination of the two molecules through one or more types of chemical bonds, through complementary base pairing, or through hydrogen bond formation.
  • bases in a probe may be joined by a linkage other than a phosphodiester bond, so long as it does not prevent hybridization.
  • oligonucleotide probes may have constituent bases joined by peptide bonds rather than phosphodiester linkages.
  • the term complementary refers to nucleobases that may hybridize to each other. For example, adenine is complementary to thymine and cytosine is complementary to guanine.
  • Hybridizations are carried out in the same solution with the following modifications: 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 ⁇ g/ml salmon sperm DNA, 10% (wt/vol) dextran sulfate, and 5-20 ⁇ 10 6 cpm of 32 P-labeled probe is used. Filters are incubated in hybridization mixture for 18-20 h at 40° C., and then washed for 1.5 h at 55° C.
  • Filters are incubated in hybridization mixture for 30 h at 50° C., and then washed for 1.5 h at 55° C. In a solution containing 2 ⁇ SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS. The wash solution is replaced with fresh solution and incubated an additional 1.5 h at 60° C. Filters are blotted dry and exposed for autoradiography.
  • defined conditions of “high stringency” are as follows. Prehybridization of filters containing DNA is carried out for 8 h to overnight at 65° C. in buffer composed of 6 ⁇ SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 ⁇ g/ml denatured salmon sperm DNA. Filters are hybridized for 48 h at 65° C. in the prehybridization mixture containing 100 ⁇ g/ml denatured salmon sperm DNA and 5-20 ⁇ 10 6 cpm of 32 P-labeled probe. Washing of filters is done at 37° C. for 1 h in a solution containing 2 ⁇ SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA. This is followed by a wash in 0.1 ⁇ SSC at 50° C. for 45 minutes.
  • the probes comprise a detectable label.
  • a detectable label refers to moieties that can be attached directly or indirectly to an oligomer or polymer to thereby render the oligomer or polymer detectable by an instrument or method.
  • a detectable label may be directly attached to a probe.
  • a detectable label may be indirectly attached to a probe.
  • a detectable label may be attached to a probe by using a linker.
  • a detectable label may be, for example, a fluorochrome, a chromophore, a spin label, a radioisotope, an enzyme, a hapten, Quantum Dot, beads, aminohexyl, pyrene, and a chemiluminescence compound, such as acridinione.
  • Fluorochromes that may be used in the method of the present invention include, but are not limited to, IR dyes, Dyomics dyes, phycoerythrine, cascade blue, Oregon green 488, pacific blue, rhodamine green, 5(6)-carboxyfluorescein, cyanine dyes (i.e., Cy2, Cy3, Cy 3.5, Cy5, Cy5.5, Cy 7) (diethyl-amino)coumarin, fluorescein (i.e., FITC), tetramethylrhodamine, lissamine, Texas Red, AMCA, TRITC, and Alexa dyes.
  • IR dyes i.e., Dyomics dyes
  • phycoerythrine phycoerythrine
  • cascade blue Oregon green 488
  • pacific blue pacific blue
  • rhodamine green 5(6)-carboxyfluorescein
  • cyanine dyes i.e., Cy2, Cy3, Cy 3.5, Cy5, Cy5.5
  • the probes of the invention may constitute part of a kit.
  • the kit is comprised of (a) at least two of the probes of the invention and (b) other reagents and compositions for performing an assay to detect a change in chromosomal structure.
  • the invention also encompasses methods for using the probes of the invention. For example, in methods for detecting a change in chromosomal structure.
  • the method comprises (a) obtaining a preparation of chromosomes; (b)contacting the preparation of chromosomes with at least two of the probes of the invention, wherein the probes each contain a different label distinguishable from the other label and wherein the labeled probes create a pattern of staining in a sample of normal control chromosomes; and (c) detecting the change in chromosomal structure by detecting a change in the pattern of staining in the chromosome preparation as compared to the normal control sample.
  • a euploidy is the condition of having a normal number of structurally normal chromosomes. For example, somatic euploid cells from a human female contain 44 autosomal chromosomes and two X-chromosomes for a total of 46 chromosomes or 23 chromosomal pairs. Euploid bulls have 58 autosomal chromosomes, one X-chromosome, and one Y-chromosome.
  • Non-exhaustive examples of chromosomal alterations include aneuploidy, gene amplifications, deletions including gene deletions, gene fusions, translocations, duplications, insertions, or inversions.
  • aneuploidy refers to any deviation from the normal euploid state or the condition of having less than or more than the normal diploid number of chromosomes. Aneuploidy is the most frequently observed type of cytogenetic abnormality. Generally, aneuploidy is recognized as a small deviation from euploidy because major deviations are rarely detected because of the lethal nature of major changes in chromosome number.
  • an amplification refers to an increase in the number of copies of a specific DNA fragment.
  • DNA fragments include, for example, a gene or an entire chromosome.
  • a deletion refers to a genetic event in which a nucleic acid sequence has been removed from a chromosome.
  • a gene fusion refers to an accidental joining of the DNA of two genes. Gene fusions may occur by translocations or inversions. Gene fusions may give rise to hybrid proteins or the misregulation of the transcription of one gene due to the juxtaposition of cis regulatory elements (e.g., enhancers or promoters) of another gene.
  • a translocation refers to a genetic event in which a part of the nucleic acid sequence of one chromosome is removed from that chromosome and attached to a different chromosome.
  • a duplication refers to the repetition of a nucleotide sequence in a chromosome or a chromosome segment. For example, a duplication may result in the repetition of a nucleotide sequence in linear juxtaposition to the duplicated sequence.
  • an insertion refers to a genetic event in which a nucleic acid sequence has been introduced between two points in a chromosome.
  • an inversion is a genetic event in which a nucleic acid sequence's orientation in a chromosome has been reversed.
  • a chromosomal breakpoint refers to a location in the chromosome where the chromosome breaks into two pieces.
  • a preparation of chromosomes refers to a composition comprised of chromosomes from a cell type of interest.
  • a cell type of interest may be a mammalian cell.
  • chromosome preparations may be produced from mammalian cells in various stages of mitosis.
  • chromosomal preparations may be produced from cells in metaphase.
  • FISH may also be used on a preparation of chromosomes from cells in interphase. Methods for producing a chromosomal preparation and nuclei for use in FISH analysis are well known to those of skill in the art.
  • the phrases “chosen from one or more of,” “chosen from at least one of,” “chosen from one or more,” and “chosen from at least one” followed by a list of items such as A, B, and C indicate that one or more of A, B, and C may be selected (e.g. only A; only B; only C; only A and B; only B and C; only A and C; or A, B, and C), and also indicate that one or more types within each A, B, or C category may be selected (e.g. only A1 and A2; only A1 and A2 and B1; only B1 and B2 and C1, etc.).
  • the probe precursor DNA was directly labeled with a fluorochrome by nick translation.
  • 10 ⁇ g of DNA was resuspended in 250 ⁇ l of reaction buffer.
  • the reaction buffer contained 25 ⁇ l 10 ⁇ nick translation buffer (500 mmol/L Tris-HCl, 100 mmol/L MgCl 2 , 1 mmol/L DTT, 100 mg/L BSA, pH 7.5); 25 ⁇ l 10 ⁇ dNTP mix (50 mmol/L Tris-HCl, 10 mmol/L EDTA, 0.5 mmol/L dATP, 0.5 mmol/L dGTP, 0.5 mmol/L dCTP, 0.34 mmol/L dTTP, pH 7.6); 4 ⁇ l of dUTP fluorochrome (1 mmol/L); 10 ⁇ l DNA polymerase I (Invitrogen, 10 ⁇ l); and 0.3 ⁇ l DNase I (Sigma Aldrich).
  • the labeling reaction was incubated for 4 hours at 15° C. The reaction was stopped by adding 25 ⁇ l 500 mmol/L EDTA and incubating for 10 minutes at 65° C. Unincorporated nucleotides were removed by centrifugation using Microcon YM-10 Centrifugal Filter Devices (Milipore). The purified labeled probe was resuspended in 25 ⁇ l of ice cold ( ⁇ 20° C.)TE buffer (10 mmol/L Tris HCl, 0.1 mmol/L EDTA, pH 8.0).
  • a slide containing metaphase spreads and interphase nuclei was pretreated in TBS (50 mmol/L Tris, 150 mmol/L NaCl, pH 7.6) with 3.7% formaldehyde for 2 minutes at room temperature, as described in Human Cytogenetics, a Practical Approach, Volume 1, 2 nd ed., D. E. Rooney and B. H. Czepulkowski editors (1992).
  • the slide was then rinsed twice in phosphate buffered saline (PBS) for 5 minutes per wash at room temperature. After rinsing, the slide was dehydrated in a cold (4° C.) series of ethanol by incubating the slide in 70% ethanol at 4° C. for 2 minutes; incubating the slide in 85% ethanol at 4° C. for 2 minutes; and then incubating the slide in 96% ethanol at 4° C. for 2 minutes, followed by air drying.
  • PBS phosphate buffered saline
  • DNA Hybridization Buffer 100 ng fluorescein labelled probe, 100 ng Texas Red labelled probe, 5 ⁇ M PNA Oligo Mix, 45% formamide, 300 mM NaCl, 5 mM NaPO4, 10% Dextran sulphate
  • 10 ⁇ L of DNA Hybridization Buffer 100 ng fluorescein labelled probe, 100 ng Texas Red labelled probe, 5 ⁇ M PNA Oligo Mix, 45% formamide, 300 mM NaCl, 5 mM NaPO4, 10% Dextran sulphate
  • the edges of the coverslip were sealed with rubber cement before denaturation at 82° C. for 5 minutes.
  • the slide was hybridized overnight at 45° C. After hybridization, the coverslip was removed and the slide was washed for 10 minutes in a stringent wash buffer (0.2 ⁇ SSC, 0.1% Triton X-100) at 65° C.
  • the slide was then rinsed in TBS for 1 minute before dehydrated in a cold (4° C.) series of 70%, 85% and 96% ethanol as described above.
  • Each slide was mounted with 10 ⁇ L of anti-fade solution (Vectashield H-1000, Vector Laboratories, Inc. Burlingame) supplemented with 0.1 ⁇ g/mL 4,6-diamidino-2-phenylindole (DAPI, Sigma Chemicals) and sealed with a coverslip.
  • the slide was analyzed using a fluorescence microscope equipped with a CCD digital camera.
  • FIGS. 2-6 provide results with probes that bind to the BCR, ETV6, MLL, TCF3, and TLX3 genes respectively.
  • BCL2 probes were hybridized to metaphase chromosomes from metaphase spreads from normal peripheral blood.
  • Two normal BCL2 loci on chromosomes 18 were represented by two yellow dots or co-localized green/red signals.
  • BCL3 probes were hybridized to metaphase chromosomes from metaphase spreads from normal peripheral blood.
  • Two normal BCL3 loci on chromosomes 19 were represented by two yellow dots or co-localized green/red signals.
  • BCL6 probes were hybridized to metaphase chromosomes from metaphase spreads from normal peripheral blood.
  • Two normal BCL6 loci on chromosomes 3 were represented by two yellow dots or co-localized green/red signals.
  • CCND1 probes were hybridized to metaphase chromosomes from metaphase spreads from normal peripheral blood. Two normal CCND1 loci on chromosomes 11 were represented by two yellow dots or co-localized green/red signals.
  • MYC probes were hybridized to metaphase chromosomes from metaphase spreads from normal peripheral blood. Two normal MYC loci on chromosomes 8 were represented by two yellow dots or co-localized green/red signals.
  • MALT probes were hybridized to metaphase chromosomes from metaphase spreads from normal-peripheral blood. Two normal MALT loci on chromosomes 18 were represented by two yellow dots or co-localized green/red signals.
  • PAX5 probes were hybridized to metaphase chromosomes from metaphase spreads from normal peripheral blood. Two normal PAX5 loci on chromosomes 9 were represented by two yellow dots or co-localized green/red signals.
  • TLX1 probes were hybridized to metaphase chromosomes from metaphase spreads from normal peripheral blood. Two normal TLX1 loci on chromosomes 10 were represented by two yellow dots or co-localized green/red signals.
  • PDGFRA probes were hybridized to metaphase chromosomes of metaphase spreads and nuclei from the cell line ELO-1, and to nuclei from normal tissue.
  • one normal PDGFRA locus on chromosome 4 was represented by a yellow dot or co-locolized green/red signals. Lack of one red signal indicated a sub-deletion.
  • An additional chromosome 4 could be detected as a result of an amplification of the derivative chromosome 4.
  • On the nuclei one normal PDGFRA locus on chromosome 4 was represented by a yellow dot or co-locolized green/red signals. Lack of one red signal indicated an sub-deletion.
  • An additional chromosome 4 could be detected as a result of an amplification of the derivative chromosome 4.
  • PDGFRB probes were also hybridized to metaphase chromosomes from metaphase spreads from the cell line NALM-6.
  • One normal PDGFRA locus on chromosome 5 was represented by a yellow dot or co-locolized green/red signals. Lack of one green signal indicated an deletion of the derivative chromosome 5, while a red signal indicated a translocation to another chromsome.

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Abstract

The present invention relates to probes for use in various applications. For example, these probes may be used to detect, quantify, identify, or analyze nucleic acid molecules or other molecules that bind to the probes. The invention also encompasses compositions and kits containing the probes and methods of using the probes.

Description

  • This application claims priority to U.S. Provisional Patent Application Nos. 60/567,570 and 60/567,440, both of which were filed May 4, 2004, and which are incorporated herein by reference in their entirety.
  • The instant application contains a “lengthy” Sequence Listing which has been submitted via CD-R in lieu of a printed paper copy, and is hereby incorporated by reference in its entirety. Said CD-R, recorded on May 3, 2005, are labeled CRF, “Copy 1” and “Copy 2”, respectively, and each contains only one identical 21.4 MB file (91386us.APP).
  • The present invention relates to probes for use in various applications. For example, these probes may be used to detect, quantify, identify, or analyze nucleic acid molecules or other molecules that bind to the probes. The invention also encompasses compositions and kits containing the probes and methods of using the probes.
  • Probe-based assays are useful in the detection, quantitation and analysis of nucleic acids by hybridizing a probe to a nucleic acid sample. Probes have long been used to analyze samples for the presence of nucleic acid from a bacteria, fungi, virus or other organism (See for example; U.S. Pat. Nos. 4,851,330, 5,288,611, 5,567,587, 5,601,984 and 5,612,183). Probe-based assays are also useful in examining genetically-based clinical conditions of interest, as discussed below.
  • There are several types of probes that may be used for hybridizing to a nucleic acid sample (See generally Szeles, Acta Microbiol. Immunol. Hungarica, 49:69-80 (2002)). These probes include short sequences of genomic DNA or cDNA, whole chromosome paints, chromosome repeats, and whole genomes. In the case of genomic probes, frequently repeated sequences in mammalian genomes have relatively little evolutionary conservation. Thus, total nuclear or genomic DNA can be used as a species-specific probe. Chromosome paints are collections of DNA sequences derived from a single chromosome type and can identify that specific chromosome type in metaphase and interphase nuclei. Different chromosomal types also have unique repeated sequences that may be targeted for probe hybridization (See Cremer et al., Hum. Genet., 74:346-52 (1986)). These chromosomal repeat probes have been cloned for more than two thirds of the human chromosome types (See Szeles, Acta Microbiol. Immunol. Hungarica, 49:69-80 (2002)). Large insert probes that target unique single-copy sequences are another example of a probe type that may be used in hybridization assays. These probes may be in cosmids, bacterial artificial chromosomes (BACs), P1 diverted artificial chromosomes (PACs), or yeast artificial chromosomes (YACs). With these large probes, the hybridization efficiency is inversely proportional to the probe size. Nonetheless, probes as small as 2 kb have been used (See Id.).
  • Detection of nucleic acid sequences by in situ hybridization, which involves a fundamental process in molecular biology, has been applied for several years (Speel, Histochem. Cell Biol., 112:89-113 (1999)). Sequence differences as subtle as a single base (point mutation) in very short oligomers (<10 base pairs “bp”) can be sufficient to enable the discrimination of the hybridization to complementary nucleic acid target sequences as compared with non-target sequences. Nonetheless, probes of greater than 10 bp in length are generally required to obtain the sequence diversity necessary to correctly identify a unique organism or clinical condition of interest. The ability to discriminate between closely related sequences is inversely proportional to the length of the hybridization probe because the difference in thermal stability decreases between wild type and mutant complexes as the probe length increases. In situ hybridization began by using probes that were radioactively labeled. Currently, however, several alternate detectable labels are available, as discussed below.
  • Fluorescence in-situ hybridization (“FISH”) is one example of a probe-based assay in which the probes of the invention may be used. FISH was initially developed in the late 1970s (Rudkin and Stollar, Nature, 265:472-73 (1977)). The technique generally entails preparing a cytological sample, labeling probes, denaturing target chromosomes and the probe, hybridizing the probe to the target sequence, and detecting a signal. Typically, the hybridization reaction fluorescently stains the target sequences so that their location, size, or number can be determined using fluorescence microscopy, flow cytometry or other suitable instrumentation. In mammalian cells, chromosomes are arranged in matching pairs. Each chromosome in the pair is a sister chromosome. DNA sequences ranging from whole genomes down to several kilobases can be studied using current hybridization techniques in combination with commercially available instrumentation.
  • FISH-based staining is sufficiently distinct such that the hybridization signals can be seen both in metaphase spreads and in interphase nuclei. Single and multicolor FISH, using probes, have been applied to different clinical applications generally known as molecular cytogenetics, including prenatal diagnosis, leukemia diagnosis, and tumor cytogenetics. In general, the FISH technique has several advantages including ease of use, rapid results, reduced background in comparison with the radioactive labels that preceded fluorescent labels, and high sensitivity.
  • The FISH technique has several applications in the clinical setting. These applications include, for example, detection of chromosomal aneuploidy in prenatal diagnoses, hematological cancers, and solid tumors; detection of gene abnormalities such as oncogene amplifications, gene deletions, or gene fusions; chromosomal structural abnormalities such as translocations, duplications, insertions, or inversions; detection of contiguous gene syndromes such as microdeletion syndrome; monitoring the genetic effect of therapy; detection of viral nucleic acids in somatic cells and viral integration sites in chromosomes; gene mapping; and cell cycle analysis (Luke and Shepelsky, Cell Vision 5:49-53 (1998)).
  • With regard to applications in cancer research, FISH analysis has lead to the identification of several deletion-prone regions in human chromosomes that have been correlated with cancers in various tissues including breast, kidney, lung, uterus, testis, and ovaries (Szeles, Acta Microbiol. Immunol. Hungarica, 49:69-80 (2002)). FISH analysis has also been used to analyze tumor-suppressor genes and DNA repair genes.
  • There are several variations of the FISH technique those in the art have used to analyze chromosomes (Luke and Shepelsky, Cell Vision 5:49-53 (1998)). For example, in comparative genomic hybridization (CGH) whole genomes are stained and compared to normal reference genomes for the detection of regions with aberrant copy number. FISH is ideally suited for the simultaneous detection of multiple hybridization probes because of the availability of spectrally distinct fluorochromes. In addition, FISH allows the accurate quantitation of hybridization signals. These characteristics paved the way for multicolor applications, which evolved to the simultaneous hybridization of 24 or even more DNA probes for the FISH-based karyotyping chromosomes. For such applications, several techniques, including multiplex FISH (m-FISH; Speicher et al., Nat. Genet., 12:368-75 (1996)), spectral karyotyping (SKY; Schröck et al., Science, 273:494-97 (1996)), combined binary ration labeling (COBRA; Tanke et al., Eur. J. Hum. Genet., 7:2-11 (1999)), or color-changing karyotyping (Henegariu et al., Nat. Genet., 23:263-64 (1999)) have been developed. Moreover, FISH-based banding technologies, such as cross-species color banding (Müller et al., Hum. Genet., 100:271-78 (1997)) or high resolution multicolor banding (Chudoba et al., Cytogenet. Cell Genet., 84:156-60 (1999)) were developed for karyotyping and to facilitate the identification of intrachromosomal rearrangements. Thus, a variety of different multicolor-FISH technologies are available to the skilled artisan. The basic principles of multicolor-FISH were reviewed recently (Lichter, Trends Genet., 13:475-79 (1997)). When used on abnormal material, the probes will stain the aberrant chromosomes thereby deducing the normal chromosomes from which they are derived (Macville M et al., Histochem Cell Biol., 108:299-305 (1997)). Specific DNA sequences, such as the ABL gene, can be reliably stained using probes of only 15 kb (Tkachuk et al., Science, 250: 559-62 (1990)). Telomeric multiplex FISH (TM-FISH) allows for simultaneous hybridization of several subtelomeric probes on one sample slide (Henegariu et al., Lab. Invest., 81:483-91 (2001)). These subtelomeric probes are between 100 kb and 1 Mb from the end of the chromosomes.
  • SKY uses charge coupled device (CCD) imaging and Fourier spectroscopy to assess the spectrum of fluorescent wavelengths for each pixel scanned, assigning a specific pseudocolor depending on the spectrum identified (Schrock et al. Science, 273:494-497 (1996)). See also the E.C.A. Newsletter for a general description of SKY, available at http://www.biologia.uniba.it/eca/NEWSLETTERS/NS-7/02-Multiplex-FISH.html. In cases of chromosomal aberrations involving small genomic segments, SKY analysis may require supplemental FISH analysis using smaller, subtelomeric probes or chromosome painting probes, (Fan et al., Genetic Testing, 4:9-14 (2000); Hilgenfeld et al., British J. Haematol., 113:305-17 (2001)).
  • Split-signal FISH (ssFISH) detects changes in chromosomal structure by using two probes, each of which is labeled by a different detectable label. The detectable labels should be distinguishable from one another. Each probe binds to the chromosome on either side of a suspected breakpoint in the chromosome. In a normal chromosome, the two probes will be proximal enough to each other such that the combined signal of their different labels forms a signal that is different from each label alone. Thus, a normal chromosomal sample will contain only the combined or fused signal of the two probes on the sister chromosomes. In an abnormal sample, where one sister chromosome has broken at the suspected break point, the fused signal will remain on the normal sister chromosome. On the broken chromosome, one probe migrates to a completely different location, where the individual signal of that translocated probe becomes apparent. The other individual probe remains on the split chromosome and, because it is no longer proximal to the other probe, emits its individual signal as well. In sum, because of the break in chromosomal structure, the two probes are no longer juxtaposed, allowing the fused signal they cause together to split into the individual signals for each probe. See generally WO 98/51817.
  • Fusion-signal FISH is similar to ssFISH in that two probes with two different, distinguishable labels are used such that proximity of the two labels produces a new fused signal. The two methods differ in that for fusion-signal FISH, the two probes bind to two different locations that are suspected to become proximal to each other as a result of a chromosomal translocation. Thus, in a normal sample, only signals from the individual probes are present and no fused signal appears. In an abnormal sample, where a piece of one chromosome has attached to another chromosome, the normal chromosomes in each of the two pairs involved will still emit each of the individual probe signals. In the abnormal chromosome, in which the probes are now proximal due to the translocation, the fused signal appears. For a general discussion of fusion-signal FISH, see Arnoldus et al., Cytogenet. Cell Genet., 54:108-11 (1990). Similar results can be obtained without the use of fluorescent microscopy. Such methods include in situ hybridization (ISH) and radioactive in situ hybridization or chromogenic in situ hybridization (CISH). See Tanner et al., Am. J. Pathol., 157:1467-72 (2000).
  • The invention provides a series of probes that may be used in the above-described assays or in other probe-based assays. For a general discussion of exemplary alternate probe-based assays see Veltman et al., Biotechniques, 35:1066-70 (2003).
    • SUMMARY OF THE INVENTION
  • The invention relates to probes that may be used in a variety of probe-based assays. In one embodiment, the probes are identical to the nucleic acid sequences set forth in SEQ ID NOS:1-107. In another embodiment, the probes are substantially identical to the nucleic acid sequences set forth in SEQ ID NOS:1-107. In yet another embodiment, the probes are labeled with a detectable label. In another embodiment, the probes are not labeled.
  • In another embodiment, the probe flanks the breakpoint region of a chromosome. In another embodiment, the probe partially overlaps the breakpoint region of a chromosome. In another embodiment, the probe extends past the breakpoint region of a chromosome.
  • In yet another embodiment, unlabeled probes are used to remove complementary labeled probes that are at a chromosomal breakpoint, thus producing labeled break point probes. For example, two differentially labeled probes extending into a chromosome breakpoint region may overlap each other. As shown in FIG. 1, when these labeled probes are used in an assay to detect a translocation, they may mask the presence of the translocation due to this overlap in probe sequence. Thus, where the differentially labeled probes should give two distinguishable signals for a translocation, the overlapping probes may instead give a signal that combines the two labels. A combined signal may falsely indicate a duplication of chromosomal sequence, rather than the actual translocation that is present in the chromosome. This problem can be solved by adding an excess amount of a “blocking probe,” which competes with and blocks the labeled probes' binding to the breakpoint region, thereby generating the correct signal. See FIG. 1. Examples of blocking probes include, but are not limited to, a nucleic acid sequence comprising a sequence that is complementary to the labeled probes, a nucleic acid sequence that binds to the labeled probes under low stringency conditions, or a nucleic acid analog probe that binds to the labeled probes under low stringency conditions. A blocking probe may be prepared from various vectors including, but not limited to, a plasmid, a cosmid, a PAC, a BAC, or a YAC. A blocking probe may be unlabeled or may comprise a detectable labeled.
  • In a further embodiment, the nucleic acid probe is selected from (a) a nucleic acid comprising the nucleotide sequence substantially as set out in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ. ID NO. 22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ-ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, or SEQ ID NO:107; and (b) a nucleic acid that hybridizes to the complement of the nucleic acid of (a) under low stringency conditions. In alternate embodiments, hybridization may occur under moderate or high stringency conditions.
  • In one embodiment, the nucleic acid probe is a fragment of the nucleic acid probes disclosed herein that is complementary to the binding area of interest.
  • In an additional embodiment, the probes may be used to detect a change in chromosomal structure. These changes in chromosomal structure may be associated with a variety of diseases. For example, lymphomas are frequently associated with translocations. As another example, the human ETV6 gene (Golub et al., Cell, 77:307-16 (1994)), also known as TEL, consists of 8 exons occupying a region of 240 kb on chromosome 12 band p13 (Baens et al., Genome Res., 6:404-13 (1996)). The ETV6 protein contains two critical domains, the 5 helix-loop-helix (HLH) dimerization domain coded by exon 3 and 4, whereas exons 6-8 code a 3′ E26 transformations-specific (ETS) DNA-binding domain (Id.). The ETV6 gene encodes a sequence-specific transcriptional repressor that is required specifically for hematopoiesis within the bone marrow (Wang et al., Genes Dev., 12:2392-402 (1998)). This gene has been identified in fusion with many different partners and in a wide spectrum of human leukemias such as in chronic myelomonocytic leukemia (CMML), acute myeloid leukemia (AML), and acute lymphoblastic leukemia (ALL) (Mitelman et al., available at http://cgap.nci.nih.gov/Chromosomes/Mitelman).
  • The most common gene rearrangement in cancer of childhood is the fusion of ETV6 and AML1 (also know as RUNX1 and CBFA2) in approximately 22-25% of the cases with childhood B-lineage ALL (Shurtleff et al., Leukemia, 9:1985-89 (1995); Romana et al., Blood, 86:4263-69 (1995)). The ETV6/AML 1 translocation involves the ETV6 disrupting the gene between the HLH motif and the ETS DNA-binding domain. This fusion results in a cryptic t(12;21) translocation that is easily missed by conventional cytogenetic analysis because the rearranged fragments barely affect the morphology of the involved chromosomes (Ferrando and Look, Sem. Hematol., 37:381-95 (2000)). Deletion of the nontranslocated ETV6 allele are frequently associated with a t(12,21) in childhood ALL (Raynaud et al., Blood, 87:2891-99 (1996); Cave et al., Leukemia, 11:1459-64 (1997)). The extent of this deletion is highly variable ranging from, in the majority of cases, where the whole of ETV6 is deleted to a small number where the deletion is intragenic (Raynaud et al., Blood, 87:2891-99 (1996)). The presence of t(12;21) is shown to be a significant favorable prognostic factor, with event-free survivals of 91% in childhood ALL (Rubnitz et al., J. Clin. Oncol., 15:1150-57 (1997)). Table 1 provides a non-exhaustive list of some of the diseases detected by the probes of the invention.
    TABLE 1
    ETV6 TCF3 TLX3 MLL BCR BCL2 BCL3
    chronic myelomonocytic leukemia (CMML) + − + + − − −
    myelodysplastic syndrome (MDS) + − − + − − −
    acute lymphoblastic leukemia (ALL) + + + + + − −
    acute myelocytic leukemia (AML) + − − + + − −
    chronic myelogenous leukemia (CML) + − − + + − −
    T-cell prolymphocytic leukemia − − − − − − −
    T-cell large granular lymphocyte leukemia (T-LGL) − − − + − − −
    Hypereosinophilic syndrome (HES) − − − − − − −
    Anaplastic large cell lymphoma (ALCL) − − − − − − +
    Lymphoplasmocytic lymphoma (LPL) − − − − − − −
    Burkitt's lymphoma (BL) − − − − − − −
    Mantle cell lymphoma (SMZL) − − − − − − −
    Splenic marginal zone lymphoma (SMZL) − − − − − − −
    Marginal zone (MALT) − − − − − − −
    Follicular lymphoma (FL) − − − − − + −
    Diffuse large cell lymphoma (DLBCL) − − − − − + −
    T-cell leukemia/lymphoma (TLL) − − − − − − −
    Multiple myeloma (MM) − − − − − + −
    lymphoma − − − − − + +
  • BCL6 BCL10 MALT1 MYC PAX5 CCND1
    chronic myelomonocytic leukemia (CMML) − − − + − −
    myelodysplastic syndrome (MDS) − − − + − −
    acute lymphoblastic leukemia (ALL) − − − + − −
    acute myelocytic leukemia (AML) − − − + − −
    chronic myelogenous leukemia (CML) − − − − − −
    T-cell prolymphocytic leukemia − − − − − −
    T-cell large granular lymphocyte leukemia (T-LGL) − − − − − −
    Hypereosinophilic syndrome (HES) − − − − − −
    Anaplastic large cell lymphoma (ALCL) − − − − − −
    Lymphoplasmocytic lymphoma (LPL) − − − − + −
    Burkitt's lymphoma (BL) − − − + − −
    Mantle cell lymphoma (SMZL) − − − − − +
    Splenic marginal zone lymphoma (SMZL) − − − − − +
    Marginal zone (MALT) − + + − − −
    Follicular lymphoma (FL) + − − − − −
    Diffuse large cell lymphoma (DLBCL) + + − + − −
    T-cell leukemia/lymphoma (TLL) − − − − − −
    Multiple myeloma (MM) − − − + − +
    lymphoma + + + + + +
  • TLX1 TCRAD TCRB TCRG PDGFRA PDGFRB
    chronic myelomonocytic leukemia (CMML) − − − − − −
    myelodysplastic syndrome (MDS) − − − − − −
    acute lymphoblastic leukemia (ALL) + − − − − −
    acute myelocytic leukemia (AML) − − − − − −
    chronic myelogenous leukemia (CML) − − − − − +
    T-cell prolymphocytic leukemia − + + + − −
    T-cell large granular lymphocyte leukemia (T-LGL) − + + + − −
    Hypereosinophilic syndrome (HES) − − − − + −
    Anaplastic large cell lymphoma (ALCL) − − − − − −
    Lymphoplasmocytic lymphoma (LPL) − − − − − −
    Burkitt's lymphoma (BL) − − − − − −
    Mantle cell lymphoma (SMZL) − − − − − −
    Splenic marginal zone lymphoma (SMZL) − − − − − −
    Marginal zone (MALT) − − − − − −
    Follicular lymphoma (FL) − − − − − −
    Diffuse large cell lymphoma (DLBCL) − − − − − −
    T-cell leukemia/lymphoma (TLL) − + + + − −
    Multiple myeloma (MM) − − − − − −
    lymphoma − + + + − −
  • Other embodiments include kits or compositions containing the probes of the invention wherein the kit or composition may be used to diagnose the aforementioned diseases or any other disease also associated with the target sequences bound by the claimed probes.
  • Additional objects and advantages of the invention will be set forth in part in the description which follows. In addition, the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.
    • BRIEF DESCRIPTION OF THE DRAWINGS
  • FIG. 1 shows using an unlabeled blocking probe to block the binding of labeled probes to a chromosome breakpoint. In the top portion of the figure, two “red probes” are depicted at the top left using thin lines, four “green probes” are depicted at the top right using thick lines. The “blocking probe” is depicted using medium-weight lines. On the three plates shown, lighter shaded areas represent the signal from the first label (for example, red), darker shaded areas represent the signal from the second label (for example, green), and unshaded areas represent co-localized (for example, green/red) signals.
  • FIG. 2 shows hybridization of BCR probes to metaphase chromosomes. (A) Drawing of the area covered by the probes, with different colored probes denoted by short and long dashed lines; (B) Hybridization of the BCR probes to metaphase spreads from normal peripheral blood. Two normal BCR loci on chromosomes 22 are represented by two light colored dots (e.g. yellow, from the superimposition of green and red signals) or co-localized different colored signals (e.g. green and red); (C) Hybridization of the BCR probes to nuclei from normal peripheral blood. Two normal BCR loci are represented by two light colored dots or co-localized different colored signals; (D) Hybridization of the BCR probes to metaphase spreads from the cell line BV173. One normal BCR locus on chromosome 22 is represented by a light colored dot or co-locolized signals from two differently colored probes (see the bright dots at the top left of the figure). Two derivative chromosomes resulting from a translocation between chromosome 9 and 22 (in cell line BV173) can be detected. An additional chromosome 9 can be detected as a result of an amplification of the derivative chromosome 9; (E) Hybridization of the BCR probes to nuclei from the cell line BV173. One normal BCR locus is represented by a light colored dot or co-locolized signals from two different colored probes. (See the brightly colored dots at the top right side of the nuclei pictured here.) Two derivative chromosomes resulting from a translocation between chromosome 9 and 22 (in cell line BV173) can be detected. An additional chromosome 9 can be detected as a result of an amplification of the derivative chromosome 9.
  • FIG. 3 shows hybridization of ETV6 probes to metaphase chromosomes. (A) Drawing of the area cover by the probes, with different colored probes denoted by short and long dashed lines; (B) Hybridization of the ETV6 probes to metaphase spreads from normal peripheral blood. Two normal ETV6 loci on chromosomes 12 are represented by two light colored dots or co-locolized different color signals; (C) Hybridization of the ETV6 probes to nuclei from normal peripheral blood. Two normal ETV6 loci are represented by two light colored dots or co-locolized different colored signals; (D) Hybridization of the ETV6 probes to metaphase spreads from the cell line REH. The normal ETV6 locus on chromosome 12 is deleted, represented by the lack of a light colored dot or co-locolized different colored signals. Instead, each signal shown on this spread is one pure color (green or red). Two derivative chromosomes resulting from a translocation between chromosome 12 and 21 (in cell line REH) can be detected. A small green signal can be detected as a remaining part of the deleted ETV6 locus; (E) Hybridization of the ETV6 probes to nuclei from the cell line REH. The normal ETV6 locus on chromosome 12 is deleted, represented by the lack of a light colored dot or co-locolized different color signals. Two derivative chromosomes resulting from a translocation between chromosome 12 and 21 (in cell line REH) can be detected. A small green signal can be detected as a remaining part of the deleted ETV6 locus (top right and bottom left dots on this spread).
  • FIG. 4 shows hybridization of MLL probes to metaphase chromosomes. (A) Drawing of the area cover by the probes, with different colored probes denoted by short and long dashed lines; (B) Hybridization of the MLL probes to metaphase spreads from normal peripheral blood. Two normal MLL loci on chromosomes 11 are represented by two light colored dots or co-locolized different colored signals (all four dots shown); (C) Hybridization of the MLL probes to nuclei from normal peripheral blood. Two normal MLL loci are represented by two light colored dots or co-locolized different colored signals (all dots shown); (D) Hybridization of the MLL probes to metaphase spreads from the cell line RS-4;11. One normal MLL locus on chromosome 11 is represented by a light colored dot or co-locolized different colored signals (at top right). Two derivative chromosomes resulting from a translocation between chromosome 4 and 11 (in cell line RS-4;11) can be detected; (E) Hybridization of the MLL probes to nuclei from the cell line RS-4;11. One normal MLL locus on chromosome 11 is represented by a light colored dot or co-locolized different colored signals (larger dots on the spread at top right and at bottom). Two derivative chromosomes resulting from a translocation between chromosome 4 and 11 (in cell line RS-4; 11) can be detected.
  • FIG. 5 shows hybridization of TCF3 probes to metaphase chromosomes. (A) Drawing of the area cover by the probes, with different colored probes denoted by short and long dashed lines; (B) Hybridization of the TCF3 probes to metaphase spreads from normal peripheral blood. Two normal TCF3 loci on chromosomes 19 are represented by two light colored dots or co-locolized different colored signals; (C) Hybridization of the TCF3 probes to nuclei from normal peripheral blood. Two normal TCF3 loci are represented by two light colored dots or co-locolized different colored signals; (D) Hybridization of the TCF3 probes to metaphase spreads from the cell line 697. One normal TCF3 locus on chromosome 19 is represented by a light colored dot or co-locolized different colored signals (two bright dots at right). Two derivative chromosomes resulting from a translocation between chromosome 1 and 19 (in cell line 697) can be detected; (E) Hybridization of the TCF3 probes to nuclei from the cell line 697. One normal TCF3 locus on chromosome 19 is represented by a light colored dot or co-locolized different colored signals (central juxtaposed dots on the spread). Two derivative chromosomes resulting from a translocation between chromosome 1 and 19 (in cell line 697) can be detected.
  • FIG. 6 shows hybridization of TLX3 probes to metaphase chromosomes. (A) Drawing of the area cover by the probes, with different colored probes denoted by short and long dashed lines; (B) Hybridization of the TLX3 probes to metaphase spreads from normal peripheral blood. Two normal TLX3 loci on chromosomes 5 are represented by two light colored dots or co-locolized different colored signals (all dots shown here); (C) Hybridization of the TLX3 probes to nuclei from normal peripheral blood. Two normal TLX3 loci are represented by two light colored dots or co-locolized different colored signals; (D) Hybridization of the TLX3 probes to metaphase spreads from the cell line HPB-ALL. One normal TLX3 locus on chromosome 5 is represented by a light colored dot or co-locolized different colored signals (at top right). Two derivative chromosomes resulting from a translocation between chromosome 5 and 14 (in cell line HPB-ALL) can be detected; (E) Hybridization of the TLX3 probes to nuclei from the cell line HPB-ALL. One normal TLX3 locus on chromosome 5 is represented by a light colored dot or co-locolized different colored signals (dot at center). Two derivative chromosomes resulting from a translocation between chromosome 5 and 14 (in cell line HPB-ALL) can be detected.
  • Color versions of FIGS. 1-6 were submitted with U.S. Provisional Application No. 60/567,570, which is incorporated herein by reference and to which this application claims priority.
    • DESCRIPTION OF THE INVENTION
  • As used herein, the term “probe” refers to a “nucleic acid” probe or to a “nucleic acid analog” probe. As used herein, the term “nucleic acid” refers to a nucleobase sequence-containing oligomer, polymer, or polymer segment, having a backbone formed solely from naturally occurring nucleotides or unmodified nucleotides. As used herein, a “nucleic acid analog” means an oligomer, polymer, or polymer segment composed of at least one modified nucleotide, or subunits derived directly from a modification of nucleotides. Non-limiting examples of naturally occurring nucleobases include: adenine, cytosine, guanine, thymine, and uracil. Non-limiting examples of modified nucleotides include: 5-propynyl-uracil, 2-thio-5-propynyl-uracil, 5-methylcytosine, pseudoisocytosine, 2-thiouracil and 2-thiothymine, 2-aminopurine, N9-(2-amino-6-chloropurine), N9-(2,6-diaminopurine), hypoxanthine, N9-(7-deaza-guanine), N9-(7-deaza-8-aza-guanine) and N8-(7-deaza-8-aza-adenine). Other non-limiting examples of suitable nucleobases include those nucleobases illustrated in FIGS. 2(A) and 2(B) of Buchardt et al. (U.S. Pat. No. 6,357,163).
  • The term “nucleic acid analog” also refers to synthetic molecules that can bind to a nucleic acid. For example, a nucleic acid analog probe may be comprised of peptide nucleic acids (PNAs), locked nucleic acids (LNAs), or any derivatized form of a nucleic acid. As used herein, the term “peptide nucleic acid” or “PNA” means any oligomer or polymer comprising at least one or more PNA subunits (residues), including, but not limited to, any of the oligomer or polymer segments referred to or claimed as peptide nucleic acids in U.S. Pat. Nos. 5,539,082, 5,527,675, 5,623,049, 5,714,331, 5,718,262, 5,736,336, 5,773,571, 5,766,855, 5,786,461, 5,837,459, 5,891,625, 5,972,610, 5,986,053, 6,107,470 6,201,103, 6,228,982 and 6,357,163; all of which are herein incorporated by reference. The term PNA also applies to any oligomer or polymer segment comprising two or more subunits of those nucleic acid mimics described in the following publications: Lagriffoul et al., Bioorganic & Medicinal Chemistry Letters, 4:1081-1082 (1994); Petersen et al., Bioorganic & Medicinal Chemistry Letters, 6: 793-796 (1996); Diderichsen et al., Tett. Lett. 37: 475-478 (1996); Fujii et al., Bioorg. Med. Chem. Lett. 7: 637-627 (1997); Jordan et al., Bioorg. Med. Chem. Lett. 7: 687-690 (1997); Krotz et al., Tett. Lett. 36: 6941-6944 (1995); Lagriffoul et al., Bioorg. Med. Chem. Lett. 4: 1081-1082 (1994); Diederichsen, U., Bioorganic & Medicinal Chemistry Letters, 7:1743-1746 (1997); Lowe et al., J. Chem. Soc. Perkin Trans. 1, (1997) 1: 539-546; Lowe et al., J. Chem. Soc. Perkin Trans. 11: 547-554 (1997); Lowe et al., J. Chem. Soc. Perkin Trans. 11:555-560 (1997); Howarth et al., J. Org. Chem. 62:5441-5450 (1997); Altmann, K-H et al., Bioorganic & Medicinal Chemistry Letters, 7:1119-1122 (1997); Diederichsen, U., Bioorganic & Med. Chem. Lett., 8:165-168 (1998); Diederichsen et al., Angew. Chem. Int. Ed., 37: 302-305 (1998); Cantin et al., Tett. Lett., 38:4211-4214 (1997); Ciapetti et al., Tetrahedron, 53: 1167-1176 (1997); Lagriffoule et al., Chem. Eur. J., 3:912-919 (1997); Kumar et al., Organic Letters 3(9): 1.269-1272 (2001); and the Peptide-Based Nucleic Acid Mimics (PENAMs) of Shah et al. as disclosed in WO96/04000. As used herein, the term “locked nucleic acid” or “LNA” means an oligomer or polymer comprising at least one or more LNA subunits. As used herein, the term “LNA subunit” means a ribonucleotide containing a methylene bridge that connects the 2′-oxygen of the ribose with the 4′-carbon. See generally, Kurreck, Eur. J. Biochem., 270:1628-44 (2003).
  • The term “identical” means that a relevant sequence is 100% identical to the non-repetitive region of a given sequence. The term “substantially identical,” or “substantially” means that a relevant sequence is at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to the non-repetitive region of a given sequence. By way of example, such sequences may be allelic variants, sequences derived from various species, or they may be derived from the given sequence by truncation, deletion, amino acid substitution or addition. For nucleic acids, the length of comparison sequences will generally be at least 50, 100, 150, 300, or more nucleotides. Percent identity between two sequences is determined by standard alignment algorithms such as, for example, Basic Local Alignment Tool (BLAST) described in Altschul et al., J. Mol. Biol., 215:403-410 (1990), the algorithm of Needleman et al., J. Mol. Biol., 48:444-453 (1970), or the algorithm of Meyers et al., Comput. Appl. Biosci., 4:11-17 (1988).
  • In one embodiment, the nucleic acid probe is selected from (a) a nucleic acid comprising at least one nucleotide sequence substantially as set out in Table 2; and (b) a nucleic acid that hybridizes to the complement of the nucleic acid of (a) under low stringency conditions.
    TABLE 2
    Probe Name SEQ ID NO
    CTD-2369H12 1
    RP11-126O16 2
    RP11-138L23 3
    RP11-317H11 4
    RP11-50C6 5
    RP11-787B21 6
    CTC-347D24 7
    CTD-3245B9 8
    RP11-770K18 9
    RP11-861M13 10
    CTD-2315O11 11
    CTD-2349A14 12
    RP11-1085P18 13
    RP11-41L5 14
    CTC-774G15 15
    CTD-2533O12 16
    RP11-359A14 17
    RP11-757G14 18
    CTD-2276B6 19
    RP11-31L9 20
    CTD-2507C12 21
    CTD-2302P22 22
    CTD-2609M2 23
    CTC-345A15 24
    RP11-21J15 25
    CTD-3050B7 26
    RP11-1147O10 27
    CTD-3235E20 28
    RP11-1152E1 29
    RP11-114C2 30
    CTD-2270P21 31
    RP11-656G2 32
    RP11-111L20 33
    RP11-110J22 34
    CTD-2375L4 35
    CTD-2568B10 36
    RP11-465M10 37
    RP11-120O8 38
    CTD-2582N5 39
    RP11-980C24 40
    RP11-440O4 41
    CTD-3232P6 42
    RP11-69M23 43
    RP11-174N16 44
    RP11-350K6 45
    CTD-2160F24 46
    CTD-2151C21 47
    CTD-2267H22 48
    RP11-1136L8 49
    CTD-2533C10 50
    RP11-382A18 51
    RP11-601F15 52
    RP11-220I1 53
    RP11-74F19 54
    RP11-344B23 55
    RP11-8N6 56
    RP11-117L21 57
    RP11-902F7 58
    RP11-1143E20 59
    RP11-156B3 60
    RP11-195M19 61
    RP11-626H12 62
    RP11-805J14 63
    RP11-21E23 64
    RP11-729E13 65
    CTD-2339D24 66
    RP11-324L3 67
    RP11-885N18 68
    RP11-654G17 69
    RP11-290M5 70
    RP11-81E15 71
    RP11-412P14 72
    RP11-114K5 73
    RP11-739N13 74
    RP11-484D4 75
    CTD-3166K20 76
    CTD-2373N7 77
    RP11-654A2 78
    RP11-246A2 79
    CTD-2355L21 80
    RP11-158G6 81
    RP11-780M2 82
    RP11-481C14 83
    CTD-2265E20 84
    RP11-790D5 85
    RP11-721D13 86
    CTD-3131N3 87
    RP11-260G10 88
    RP11-115G1 89
    CTD-3053D22 90
    RP11-426J23 91
    RP11-114B6 92
    CTD-2505P4 93
    CTD-2191C10 94
    RP11-1084E14 95
    CTD-2371J15 96
    RP11-571I18 97
    RP11-39D6 98
    CTD-3033C24 99
    RP11-545H22 100
    RP11-84L10 101
    CTD-3180G20 102
    RP11-1120I24 103
    RP11-759G10 104
    RP11-3G18 105
    RP11-136E22 106
    RP11-1079A8 107
  • In an alternate embodiment, the nucleic acid probes of the invention are identical to at least one of the sequences identified in Table 2.
  • A probe binds to a target sequence under certain conditions. The term “bind” is synonymous with “hybridize.” When two molecules hybridize, they form a combination of the two molecules through one or more types of chemical bonds, through complementary base pairing, or through hydrogen bond formation. In addition, bases in a probe may be joined by a linkage other than a phosphodiester bond, so long as it does not prevent hybridization. Thus, oligonucleotide probes may have constituent bases joined by peptide bonds rather than phosphodiester linkages. As used herein, the term complementary refers to nucleobases that may hybridize to each other. For example, adenine is complementary to thymine and cytosine is complementary to guanine. As used herein, the term “binds under certain conditions” is intended to describe conditions for hybridization and washes under which nucleotide sequences that are significantly identical or homologous to each other remain bound to each other. The conditions may be such that sequences, which are at least about 70%, such as at least about 80%, and such as at least about 85-90% identical, remain bound to each other. As used herein, the term “target sequence” refers to the nucleobase sequence sought to be determined. The nucleobase sequence can be a subsequence of a nucleic acid molecule of interest (e.g. a chromosome). In other embodiments, the target sequences are located in the ETV6 gene, the MLL gene, the TCF3 gene (also known as the E2A gene), or the TLX3 gene (also known as the HOX11L2 gene).
  • Appropriate hybridization conditions can be selected by those skilled in the art with minimal experimentation as exemplified in Ausubel et al. (1995), Current Protocols in Molecular Biology, John Wiley & Sons, sections 2, 4, and 6. Additionally, stringency conditions are described in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Press, chapters 7, 9, and 11. As used herein, defined conditions of “low stringency” are as follows. Filters containing DNA are pretreated for 6 h at 40° C. in a solution containing 35% formamide, 5×SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500 μg/ml denatured salmon sperm DNA. Hybridizations are carried out in the same solution with the following modifications: 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 μg/ml salmon sperm DNA, 10% (wt/vol) dextran sulfate, and 5-20×106 cpm of 32P-labeled probe is used. Filters are incubated in hybridization mixture for 18-20 h at 40° C., and then washed for 1.5 h at 55° C. In a solution containing 2×SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS. The wash solution is replaced with fresh solution and incubated an additional 1.5 h at 60° C. Filters are blotted dry and exposed for autoradiography.
  • As used herein, defined conditions of “moderate stringency” are as follows. Filters containing DNA are pretreated for 7 h at 50° C. in a solution containing 35% formamide, 5×SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500 μg/ml denatured salmon sperm DNA. Hybridizations are carried out in the same solution with the following modifications: 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 μg/ml salmon sperm DNA, 10% (wt/vol) dextran sulfate, and 5-20×106 cpm of 32P-labeled probe is used. Filters are incubated in hybridization mixture for 30 h at 50° C., and then washed for 1.5 h at 55° C. In a solution containing 2×SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS. The wash solution is replaced with fresh solution and incubated an additional 1.5 h at 60° C. Filters are blotted dry and exposed for autoradiography.
  • As used herein, defined conditions of “high stringency” are as follows. Prehybridization of filters containing DNA is carried out for 8 h to overnight at 65° C. in buffer composed of 6×SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 μg/ml denatured salmon sperm DNA. Filters are hybridized for 48 h at 65° C. in the prehybridization mixture containing 100 μg/ml denatured salmon sperm DNA and 5-20×106 cpm of 32P-labeled probe. Washing of filters is done at 37° C. for 1 h in a solution containing 2×SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA. This is followed by a wash in 0.1×SSC at 50° C. for 45 minutes.
  • Other conditions of low, moderate, and high stringency well known in the art (e.g., as employed for cross-species hybridizations) may be used if the above conditions are inappropriate (e.g., as employed for cross-species hybridizations).
  • In one embodiment, the probes comprise a detectable label. As used herein, a detectable label refers to moieties that can be attached directly or indirectly to an oligomer or polymer to thereby render the oligomer or polymer detectable by an instrument or method. In one embodiment, a detectable label may be directly attached to a probe. In another embodiment, a detectable label may be indirectly attached to a probe. For example, a detectable label may be attached to a probe by using a linker. A detectable label may be, for example, a fluorochrome, a chromophore, a spin label, a radioisotope, an enzyme, a hapten, Quantum Dot, beads, aminohexyl, pyrene, and a chemiluminescence compound, such as acridinione. Fluorochromes that may be used in the method of the present invention include, but are not limited to, IR dyes, Dyomics dyes, phycoerythrine, cascade blue, Oregon green 488, pacific blue, rhodamine green, 5(6)-carboxyfluorescein, cyanine dyes (i.e., Cy2, Cy3, Cy 3.5, Cy5, Cy5.5, Cy 7) (diethyl-amino)coumarin, fluorescein (i.e., FITC), tetramethylrhodamine, lissamine, Texas Red, AMCA, TRITC, and Alexa dyes. Haptens that may be used in the present invention include, but are not limited to, 5(6)-carboxyfluorescein, 2,4-dinitrophenyl, digoxigenin, rhodamine, bromodeoxy uridine, acetylaminoflurene, mercury trinitrophenol, estradiol, and biotin. Enzymes that may be used in the present invention include, but are not limited to, soybean peroxidase, alkaline phosphatase, and horseradish peroxidase.
  • The probes of the invention may constitute part of a kit. In one embodiment, the kit is comprised of (a) at least two of the probes of the invention and (b) other reagents and compositions for performing an assay to detect a change in chromosomal structure. The invention also encompasses methods for using the probes of the invention. For example, in methods for detecting a change in chromosomal structure. In another embodiment, the method comprises (a) obtaining a preparation of chromosomes; (b)contacting the preparation of chromosomes with at least two of the probes of the invention, wherein the probes each contain a different label distinguishable from the other label and wherein the labeled probes create a pattern of staining in a sample of normal control chromosomes; and (c) detecting the change in chromosomal structure by detecting a change in the pattern of staining in the chromosome preparation as compared to the normal control sample.
  • As used herein, the term “change in chromosomal structure” refers to an alteration in the chromosomes in a test sample as compared to a normal chromosome control. As used herein, a euploidy is the condition of having a normal number of structurally normal chromosomes. For example, somatic euploid cells from a human female contain 44 autosomal chromosomes and two X-chromosomes for a total of 46 chromosomes or 23 chromosomal pairs. Euploid bulls have 58 autosomal chromosomes, one X-chromosome, and one Y-chromosome.
  • Non-exhaustive examples of chromosomal alterations include aneuploidy, gene amplifications, deletions including gene deletions, gene fusions, translocations, duplications, insertions, or inversions. As used herein, aneuploidy refers to any deviation from the normal euploid state or the condition of having less than or more than the normal diploid number of chromosomes. Aneuploidy is the most frequently observed type of cytogenetic abnormality. Generally, aneuploidy is recognized as a small deviation from euploidy because major deviations are rarely detected because of the lethal nature of major changes in chromosome number. As used herein, an amplification refers to an increase in the number of copies of a specific DNA fragment. Such DNA fragments include, for example, a gene or an entire chromosome. As used herein, a deletion refers to a genetic event in which a nucleic acid sequence has been removed from a chromosome. As used herein, a gene fusion refers to an accidental joining of the DNA of two genes. Gene fusions may occur by translocations or inversions. Gene fusions may give rise to hybrid proteins or the misregulation of the transcription of one gene due to the juxtaposition of cis regulatory elements (e.g., enhancers or promoters) of another gene. As used herein, a translocation refers to a genetic event in which a part of the nucleic acid sequence of one chromosome is removed from that chromosome and attached to a different chromosome. As used herein, a duplication refers to the repetition of a nucleotide sequence in a chromosome or a chromosome segment. For example, a duplication may result in the repetition of a nucleotide sequence in linear juxtaposition to the duplicated sequence. As used herein, an insertion refers to a genetic event in which a nucleic acid sequence has been introduced between two points in a chromosome. As used herein, an inversion is a genetic event in which a nucleic acid sequence's orientation in a chromosome has been reversed. As used herein, a chromosomal breakpoint refers to a location in the chromosome where the chromosome breaks into two pieces.
  • As used herein, a preparation of chromosomes refers to a composition comprised of chromosomes from a cell type of interest. A cell type of interest may be a mammalian cell. In a further embodiment, chromosome preparations may be produced from mammalian cells in various stages of mitosis. For example, chromosomal preparations may be produced from cells in metaphase. FISH may also be used on a preparation of chromosomes from cells in interphase. Methods for producing a chromosomal preparation and nuclei for use in FISH analysis are well known to those of skill in the art.
  • As used herein, the phrases “chosen from one or more of,” “chosen from at least one of,” “chosen from one or more,” and “chosen from at least one” followed by a list of items such as A, B, and C, indicate that one or more of A, B, and C may be selected (e.g. only A; only B; only C; only A and B; only B and C; only A and C; or A, B, and C), and also indicate that one or more types within each A, B, or C category may be selected (e.g. only A1 and A2; only A1 and A2 and B1; only B1 and B2 and C1, etc.).
  • Reference will now be made in detail to the following Examples, which are provided solely to further describe the invention. These Examples are in no way intended to limit the scope or meaning of the claims.
    • EXAMPLES Example 1
  • Unique sequence probes were prepared as follows. Cells containing vectors comprising the probe of interest were grown in appropriate conditions for each cell line. DNA containing the probe sequence was isolated from the cell culture. See generally Zhao and Stodolsky, Methods in Molecular Biology: Library Construction, Physical Mapping, and Sequencing, Vol. 1, 1st ed., Humana Press (2004).
  • The probe precursor DNA was directly labeled with a fluorochrome by nick translation. On ice, 10 μg of DNA was resuspended in 250 μl of reaction buffer. The reaction buffer contained 25 μl 10× nick translation buffer (500 mmol/L Tris-HCl, 100 mmol/L MgCl2, 1 mmol/L DTT, 100 mg/L BSA, pH 7.5); 25 μl 10×dNTP mix (50 mmol/L Tris-HCl, 10 mmol/L EDTA, 0.5 mmol/L dATP, 0.5 mmol/L dGTP, 0.5 mmol/L dCTP, 0.34 mmol/L dTTP, pH 7.6); 4 μl of dUTP fluorochrome (1 mmol/L); 10 μl DNA polymerase I (Invitrogen, 10 μl); and 0.3 μl DNase I (Sigma Aldrich). The labeling reaction was incubated for 4 hours at 15° C. The reaction was stopped by adding 25 μl 500 mmol/L EDTA and incubating for 10 minutes at 65° C. Unincorporated nucleotides were removed by centrifugation using Microcon YM-10 Centrifugal Filter Devices (Milipore). The purified labeled probe was resuspended in 25 μl of ice cold (−20° C.)TE buffer (10 mmol/L Tris HCl, 0.1 mmol/L EDTA, pH 8.0).
    • Example 2
  • A slide containing metaphase spreads and interphase nuclei was pretreated in TBS (50 mmol/L Tris, 150 mmol/L NaCl, pH 7.6) with 3.7% formaldehyde for 2 minutes at room temperature, as described in Human Cytogenetics, a Practical Approach, Volume 1, 2nd ed., D. E. Rooney and B. H. Czepulkowski editors (1992). The slide was then rinsed twice in phosphate buffered saline (PBS) for 5 minutes per wash at room temperature. After rinsing, the slide was dehydrated in a cold (4° C.) series of ethanol by incubating the slide in 70% ethanol at 4° C. for 2 minutes; incubating the slide in 85% ethanol at 4° C. for 2 minutes; and then incubating the slide in 96% ethanol at 4° C. for 2 minutes, followed by air drying.
  • On each target area, 10 μL of DNA Hybridization Buffer (100 ng fluorescein labelled probe, 100 ng Texas Red labelled probe, 5 μM PNA Oligo Mix, 45% formamide, 300 mM NaCl, 5 mM NaPO4, 10% Dextran sulphate) was added and an 18×18 mm coverslip was applied to cover the hybridization area. The edges of the coverslip were sealed with rubber cement before denaturation at 82° C. for 5 minutes. The slide was hybridized overnight at 45° C. After hybridization, the coverslip was removed and the slide was washed for 10 minutes in a stringent wash buffer (0.2×SSC, 0.1% Triton X-100) at 65° C.
  • The slide was then rinsed in TBS for 1 minute before dehydrated in a cold (4° C.) series of 70%, 85% and 96% ethanol as described above. Each slide was mounted with 10 μL of anti-fade solution (Vectashield H-1000, Vector Laboratories, Inc. Burlingame) supplemented with 0.1 μg/mL 4,6-diamidino-2-phenylindole (DAPI, Sigma Chemicals) and sealed with a coverslip. The slide was analyzed using a fluorescence microscope equipped with a CCD digital camera.
  • FIGS. 2-6 provide results with probes that bind to the BCR, ETV6, MLL, TCF3, and TLX3 genes respectively.
    • Example 3
  • BCL2 probes were hybridized to metaphase chromosomes from metaphase spreads from normal peripheral blood. Two normal BCL2 loci on chromosomes 18 were represented by two yellow dots or co-localized green/red signals.
  • BCL3 probes were hybridized to metaphase chromosomes from metaphase spreads from normal peripheral blood. Two normal BCL3 loci on chromosomes 19 were represented by two yellow dots or co-localized green/red signals.
  • BCL6 probes were hybridized to metaphase chromosomes from metaphase spreads from normal peripheral blood. Two normal BCL6 loci on chromosomes 3 were represented by two yellow dots or co-localized green/red signals.
  • CCND1 probes were hybridized to metaphase chromosomes from metaphase spreads from normal peripheral blood. Two normal CCND1 loci on chromosomes 11 were represented by two yellow dots or co-localized green/red signals.
  • MYC probes were hybridized to metaphase chromosomes from metaphase spreads from normal peripheral blood. Two normal MYC loci on chromosomes 8 were represented by two yellow dots or co-localized green/red signals.
  • MALT probes were hybridized to metaphase chromosomes from metaphase spreads from normal-peripheral blood. Two normal MALT loci on chromosomes 18 were represented by two yellow dots or co-localized green/red signals.
  • PAX5 probes were hybridized to metaphase chromosomes from metaphase spreads from normal peripheral blood. Two normal PAX5 loci on chromosomes 9 were represented by two yellow dots or co-localized green/red signals.
  • TLX1 probes were hybridized to metaphase chromosomes from metaphase spreads from normal peripheral blood. Two normal TLX1 loci on chromosomes 10 were represented by two yellow dots or co-localized green/red signals.
  • PDGFRA probes were hybridized to metaphase chromosomes of metaphase spreads and nuclei from the cell line ELO-1, and to nuclei from normal tissue. On the metaphase spreads, one normal PDGFRA locus on chromosome 4 was represented by a yellow dot or co-locolized green/red signals. Lack of one red signal indicated a sub-deletion. An additional chromosome 4 could be detected as a result of an amplification of the derivative chromosome 4. On the nuclei, one normal PDGFRA locus on chromosome 4 was represented by a yellow dot or co-locolized green/red signals. Lack of one red signal indicated an sub-deletion. An additional chromosome 4 could be detected as a result of an amplification of the derivative chromosome 4.
  • PDGFRB probes were also hybridized to metaphase chromosomes from metaphase spreads from the cell line NALM-6. One normal PDGFRA locus on chromosome 5 was represented by a yellow dot or co-locolized green/red signals. Lack of one green signal indicated an deletion of the derivative chromosome 5, while a red signal indicated a translocation to another chromsome.
  • Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only. All references either discussed or referred to herein are incorporated by reference.

Claims (20)

1. An nucleic acid probe selected from:
(a) a nucleic acid comprising a nucleotide sequence substantially as set out in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ. ID NO. 22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, or SEQ ID NO:107; and (b) at least one nucleic acid or nucleic acid analog that hybridizes to the complement of the nucleic acid of (a) under low stringency conditions.
2. The nucleic acid probe of claim 1, wherein the nucleic acid of part (a) comprises a nucleotide sequence that is 70% identical to the sequence set out in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ. ID NO. 22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, or SEQ ID NO:107.
3. The nucleic acid probe of claim 1, wherein the nucleic acid of part (a) comprises a nucleotide sequence that is 80% identical to the sequence set out in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ. ID NO. 22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, or SEQ ID NO:107.
4. The nucleic acid probe of claim 1, wherein the nucleic acid of part (a) comprises a nucleotide sequence that is 90% identical to the sequence set out in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ. ID NO. 22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, or SEQ ID NO:107.
5. The nucleic acid probe of claim 1, wherein the nucleic acid of part (a) comprises a nucleotide sequence that is 95% identical to the sequence set out in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ. ID NO. 22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, or SEQ ID NO:107.
6. The nucleic acid probe of claim 1, wherein the nucleic acid of part (a) comprises a nucleotide sequence that is identical to the sequence set out in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ. ID NO. 22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, or SEQ ID NO:107.
7. The nucleic acid probe of claim 1, wherein the probe comprises at least one detectable label.
8. The nucleic acid probe of claim 1, wherein the at least one detectable label is chosen from at least one chromophore, fluorochrome, spin label, radioisotope, enzyme, hapten, Quantum Dot, bead, aminohexyl, pyrene, and chemiluminescent compound.
9. The nucleic acid probe of claim 8, wherein the fluorochrome is chosen from at least one of 5(6)-carboxyfluorescein, Cy2, Cy3; Cy 3.5, Cy5, Cy5.5, Cy 7, (diethylamino)coumarin, fluorescein, tetramethylrhodamine, lissamine, Texas Red, AMCA, TRITC, IR dyes, Dyomics dyes, phycoerythrine, cascade blue, Oregon green 488, pacific blue, rhodamine green, and Alexa dyes.
10. The nucleic acid probe of claim 8, wherein the hapten is chosen from at least one of 5(6)-carboxyfluorescein, 2,4-dinitrophenyl, digoxigenin, rhodamine, bromodeoxy uridine, sulfonate, acetylaminoflurene, mercury trintrophonol, estradiol, and biotin.
11. The nucleic acid probe of claim 8, wherein the enzyme is chosen from at least one of soybean peroxidase, alkaline phosphatase, and horseradish peroxidase.
12. The nucleic acid probe of claim 8, wherein the chemiluminescent compound is acridinione.
13. A nucleic acid probe selected from:
(a) a nucleic acid comprising a complement of a nucleotide sequence substantially as set out in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ. ID NO. 22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, or SEQ ID NO:107; and
(b) at least one nucleic acid or nucleic acid analog that hybridizes to the nucleic acid of (a) under low stringency conditions.
14. A kit comprising:
(a) at least two of the nucleic acid probes of claim 1; and
(b) reagents and compositions for performing an assay to detect a change in chromosomal structure.
15. The kit of claim 14, further comprising a reagent to block non-specific binding of the nucleic acids to a sample of chromosomes, wherein the reagent is chosen from PNA and total human DNA.
16. A method for detecting a change in chromosomal structure comprising:
a) obtaining a preparation of chromosomes;
b) contacting the preparation of chromosomes with at least two of the nucleic acid probes of claim 1, wherein each of the probes of claim 1 comprises a different label so that each nucleic acid of claim 1 is distinguishable from the other nucleic acids of claim 1; wherein the labeled nucleic acid probes create an identifiable pattern of staining upon binding to a normal chromosomal control sample; and
c) detecting a change in chromosomal structure by detecting a change in the pattern of staining in the chromosome preparation as compared to the pattern of staining in a normal chromosomal control sample.
17. A composition comprising at least one of the nucleic acid probes as set forth in claim 16 and at least one blocking probe.
18. The composition of claim 17, wherein the blocking probe is chosen from one of a nucleic acid and a nucleic acid analog.
19. The composition of claim 17, wherein the blocking probe is unlabeled.
20. The composition of claim 17, wherein the blocking probe comprises a detectable label.
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009020932A3 (en) * 2007-08-03 2009-07-09 Biocept Inc In-situ hybridization to detect rna and dna markers
WO2010097655A1 (en) 2009-02-26 2010-09-02 Dako Denmark A/S Compositions and methods for rna hybridization applications
WO2011067678A2 (en) 2009-12-02 2011-06-09 Matthiesen Steen H Compositions and methods for performing hybridizations with no denaturation
US20110229975A1 (en) * 2008-05-27 2011-09-22 Steen Hauge Matthiesen Hybridization Compositions and Methods
WO2012131673A2 (en) * 2011-03-31 2012-10-04 Hadasit Medical Research Services And Development Ltd Ccat-1 silencing nucleic acid agents for treating cancer
WO2013046033A1 (en) 2011-09-30 2013-04-04 Dako Denmark A/S Hybridization compositions and methods using formamide
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CN105483256A (en) * 2015-12-30 2016-04-13 广州安必平医药科技股份有限公司 BCR gene and ABL gene detection probe, preparation method thereof and reagent kit
US20170191133A1 (en) * 2008-05-28 2017-07-06 Genomedx Biosciences, Inc. Systems and methods for expression-based discrimination of distinct clinical disease states in prostate cancer
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US11035005B2 (en) 2012-08-16 2021-06-15 Decipher Biosciences, Inc. Cancer diagnostics using biomarkers
US11078542B2 (en) 2017-05-12 2021-08-03 Decipher Biosciences, Inc. Genetic signatures to predict prostate cancer metastasis and identify tumor aggressiveness
US11208697B2 (en) 2017-01-20 2021-12-28 Decipher Biosciences, Inc. Molecular subtyping, prognosis, and treatment of bladder cancer
US11414708B2 (en) 2016-08-24 2022-08-16 Decipher Biosciences, Inc. Use of genomic signatures to predict responsiveness of patients with prostate cancer to post-operative radiation therapy
EP4092136A1 (en) * 2021-05-20 2022-11-23 Sophia Genetics S.A. Capture probes and uses thereof
US11873532B2 (en) 2017-03-09 2024-01-16 Decipher Biosciences, Inc. Subtyping prostate cancer to predict response to hormone therapy

Families Citing this family (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP5733977B2 (en) * 2008-05-19 2015-06-10 株式会社セリッシュエフディー RNAin situ hybridization
WO2010138460A1 (en) 2009-05-26 2010-12-02 Quest Diagnostics Investments Incorporated Methods for detecting gene dysregulations
EP2487250A4 (en) * 2009-10-06 2013-03-20 Fujirebio Kk Method for detecting fusion gene
WO2011047329A2 (en) * 2009-10-15 2011-04-21 Life Technologies Corporation Novel human single nucleotide polymorphisms
WO2011079191A1 (en) 2009-12-23 2011-06-30 Quest Diagnostics Investments Incorporated Tmprss2 for the diagnosis of prostate disease
US8828688B2 (en) * 2010-05-27 2014-09-09 Affymetrix, Inc. Multiplex amplification methods
CZ2010441A3 (en) * 2010-06-04 2011-07-20 Masarykova Univerzita Detection method of chromosomal translocation t(11;14)(q13;q32) and oligonucleotides for use in this method
CN103534359B (en) 2011-03-14 2016-07-06 文塔纳医疗系统公司 Analyze method and the system thereof of chromosome translocation
DE102011100242A1 (en) 2011-05-02 2012-11-08 Zytovision Gmbh Method for detecting chromosomal aberration
EP2883964A4 (en) * 2012-08-09 2016-04-27 Japan Found Cancer Method for labelling of cleavage of nucleic acid sequence
WO2016011086A2 (en) * 2014-07-14 2016-01-21 Fry Laboratories, LLC Method and kit for protozoa characterization
DK3109324T5 (en) 2015-06-23 2019-02-11 Zytovision Gmbh PROCEDURE FOR THE DETECTION OF CHROMOSOMA DIFFERENCES
US11149299B2 (en) * 2015-06-25 2021-10-19 Ramesh Vallabhaneni Method and system for multiplex profiling of chromosomes in biological samples using target-specific DNA probes
JP6841609B2 (en) 2015-07-10 2021-03-10 ３スキャン インコーポレイテッド Spatial multiplexing of histological staining
CN106222169A (en) * 2015-12-13 2016-12-14 周平 Long-chain non-coding RNA APOC1P1-3 gene and application thereof
EP3321375A1 (en) * 2016-11-15 2018-05-16 ZytoVision GmbH Method for the detection of deletions, duplications or inversions in the same critical region
WO2023233437A1 (en) * 2022-06-01 2023-12-07 Universita' Degli Studi Di Trento Non-coding rna sequences capable of increasing the expression of chd8 and chd2 proteins

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6357163B1 (en) * 1991-05-24 2002-03-19 Ole Buchardt Use of nucleic acid analogues in diagnostics and analytical procedures

Family Cites Families (20)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU647741B2 (en) * 1989-12-01 1994-03-31 Regents Of The University Of California, The Methods and compositions for chromosome-specific staining
US5714331A (en) * 1991-05-24 1998-02-03 Buchardt, Deceased; Ole Peptide nucleic acids having enhanced binding affinity, sequence specificity and solubility
US5539082A (en) * 1993-04-26 1996-07-23 Nielsen; Peter E. Peptide nucleic acids
US5719262A (en) * 1993-11-22 1998-02-17 Buchardt, Deceased; Ole Peptide nucleic acids having amino acid side chains
US5766855A (en) * 1991-05-24 1998-06-16 Buchardt, Deceased; Ole Peptide nucleic acids having enhanced binding affinity and sequence specificity
US5641625A (en) * 1992-05-22 1997-06-24 Isis Pharmaceuticals, Inc. Cleaving double-stranded DNA with peptide nucleic acids
US6228982B1 (en) * 1992-05-22 2001-05-08 Benget Norden Double-stranded peptide nucleic acids
US6025126A (en) * 1991-10-28 2000-02-15 Arch Development Corporation Methods and compositions for the detection of chromosomal aberrations
GB9211979D0 (en) * 1992-06-05 1992-07-15 Buchard Ole Uses of nucleic acid analogues
US5527675A (en) * 1993-08-20 1996-06-18 Millipore Corporation Method for degradation and sequencing of polymers which sequentially eliminate terminal residues
DE4331012A1 (en) * 1993-09-13 1995-03-16 Bayer Ag Nucleic acid-binding oligomers with N-branching for therapy and diagnostics
GB2284209A (en) * 1993-11-25 1995-05-31 Ole Buchardt Nucleic acid analogue-induced transcription of RNA from a double-stranded DNA template
US5705333A (en) 1994-08-05 1998-01-06 The Regents Of The University Of California Peptide-based nucleic acid mimics(PENAMS)
US5750340A (en) * 1995-04-07 1998-05-12 University Of New Mexico In situ hybridization solution and process
ATE362546T1 (en) 1995-10-12 2007-06-15 Lansdorp Peter M METHOD FOR DETECTING MULTIPLE COPIES OF A REPEAT SEQUENCE IN A NUCLEIC ACID MOLECULE
US20030096255A1 (en) * 1997-02-19 2003-05-22 Felix Carolyn A. Methods and kits for analysis of chromosomal rearrangements associated with cancer
EP0878552A1 (en) * 1997-05-13 1998-11-18 Erasmus Universiteit Rotterdam Molecular detection of chromosome aberrations
US6107470A (en) * 1997-05-29 2000-08-22 Nielsen; Peter E. Histidine-containing peptide nucleic acids
US6414133B1 (en) * 1998-10-13 2002-07-02 Ventana Medical Systems, Inc. Multiple fusion probes
US7585964B2 (en) * 2001-05-14 2009-09-08 Cancer Genetics, Inc. Methods of analyzing chromosomal translocations using fluorescence in situ hybridization (FISH)

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6357163B1 (en) * 1991-05-24 2002-03-19 Ole Buchardt Use of nucleic acid analogues in diagnostics and analytical procedures

Cited By (38)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009020932A3 (en) * 2007-08-03 2009-07-09 Biocept Inc In-situ hybridization to detect rna and dna markers
US11834703B2 (en) 2008-05-27 2023-12-05 Agilent Technologies, Inc. Hybridization compositions and methods
EP3733871A1 (en) 2008-05-27 2020-11-04 Dako Denmark A/S Compositions and methods for detection of chromosomal aberrations with novel hybridization buffers
EP3360973A1 (en) 2008-05-27 2018-08-15 Dako Denmark A/S Compositions and methods for detection of chromosomal aberrations with novel hybridization buffers
US11118214B2 (en) * 2008-05-27 2021-09-14 Agilent Technologies, Inc. Hybridization compositions and methods
US20110229975A1 (en) * 2008-05-27 2011-09-22 Steen Hauge Matthiesen Hybridization Compositions and Methods
EP3219812A1 (en) 2008-05-27 2017-09-20 Dako Denmark A/S Hybridization compositions and methods
EP3138925A1 (en) 2008-05-27 2017-03-08 Dako Denmark A/S Compositions and methods for detection of chromosomal aberrations with novel hybridization buffers
US9297035B2 (en) 2008-05-27 2016-03-29 Dako Denmark A/S Compositions and methods for detection of chromosomal aberrations with novel hybridization buffers
US10865452B2 (en) * 2008-05-28 2020-12-15 Decipher Biosciences, Inc. Systems and methods for expression-based discrimination of distinct clinical disease states in prostate cancer
US20170191133A1 (en) * 2008-05-28 2017-07-06 Genomedx Biosciences, Inc. Systems and methods for expression-based discrimination of distinct clinical disease states in prostate cancer
EP3243909A1 (en) 2009-02-26 2017-11-15 Dako Denmark A/S Methods for performing hybridizations with separate denaturation of the sample and probe
WO2010097707A1 (en) 2009-02-26 2010-09-02 Dako Denmark A/S Compositions and methods for performing hybridizations with separate denaturation of the sample and probe
US9303287B2 (en) 2009-02-26 2016-04-05 Dako Denmark A/S Compositions and methods for RNA hybridization applications
US9388456B2 (en) 2009-02-26 2016-07-12 Dako Denmark A/S Compositions and methods for performing a stringent wash step in hybridization applications
WO2010097655A1 (en) 2009-02-26 2010-09-02 Dako Denmark A/S Compositions and methods for rna hybridization applications
WO2010097656A1 (en) 2009-02-26 2010-09-02 Dako Denmark A/S Compositions and methods for performing a stringent wash step in hybridization applications
US9309562B2 (en) 2009-02-26 2016-04-12 Dako Denmark A/S Compositions and methods for performing hybridizations with separate denaturation of the sample and probe
US11795499B2 (en) 2009-02-26 2023-10-24 Agilent Technologies, Inc. Compositions and methods for performing hybridizations with separate denaturation of the sample and probe
US10202638B2 (en) 2009-02-27 2019-02-12 Dako Denmark A/S Compositions and methods for performing hybridizations with separate denaturation of the sample and probe
WO2011067678A2 (en) 2009-12-02 2011-06-09 Matthiesen Steen H Compositions and methods for performing hybridizations with no denaturation
WO2012131673A2 (en) * 2011-03-31 2012-10-04 Hadasit Medical Research Services And Development Ltd Ccat-1 silencing nucleic acid agents for treating cancer
WO2012131673A3 (en) * 2011-03-31 2012-11-22 Hadasit Medical Research Services And Development Ltd Ccat-1 silencing nucleic acid agents for treating cancer
US10662465B2 (en) 2011-09-30 2020-05-26 Agilent Technologies, Inc. Hybridization compositions and methods using formamide
WO2013046033A1 (en) 2011-09-30 2013-04-04 Dako Denmark A/S Hybridization compositions and methods using formamide
WO2013057310A2 (en) 2011-10-21 2013-04-25 Dako Denmark A/S Hybridization compositions and methods
US11118226B2 (en) 2011-10-21 2021-09-14 Agilent Technologies, Inc. Hybridization compositions and methods
EP3252173A1 (en) 2011-10-21 2017-12-06 Dako Denmark A/S Hybridization compositions and methods
US11035005B2 (en) 2012-08-16 2021-06-15 Decipher Biosciences, Inc. Cancer diagnostics using biomarkers
CN105483256A (en) * 2015-12-30 2016-04-13 广州安必平医药科技股份有限公司 BCR gene and ABL gene detection probe, preparation method thereof and reagent kit
US11414708B2 (en) 2016-08-24 2022-08-16 Decipher Biosciences, Inc. Use of genomic signatures to predict responsiveness of patients with prostate cancer to post-operative radiation therapy
WO2018132755A1 (en) * 2017-01-12 2018-07-19 Duke University Compositions and methods for disrupting the molecular mechanisms associated with mitochondrial dysfunction and neurodegenerative disease
US11352625B2 (en) 2017-01-12 2022-06-07 Duke University Compositions and methods for disrupting the molecular mechanisms associated with mitochondrial dysfunction and neurodegenerative disease
US11932855B2 (en) 2017-01-12 2024-03-19 Duke University Compositions and methods for disrupting the molecular mechanisms associated with mitochondrial dysfunction and neurodegenerative disease
US11208697B2 (en) 2017-01-20 2021-12-28 Decipher Biosciences, Inc. Molecular subtyping, prognosis, and treatment of bladder cancer
US11873532B2 (en) 2017-03-09 2024-01-16 Decipher Biosciences, Inc. Subtyping prostate cancer to predict response to hormone therapy
US11078542B2 (en) 2017-05-12 2021-08-03 Decipher Biosciences, Inc. Genetic signatures to predict prostate cancer metastasis and identify tumor aggressiveness
EP4092136A1 (en) * 2021-05-20 2022-11-23 Sophia Genetics S.A. Capture probes and uses thereof

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