WO2013167387A1 - Uniquely specific probes for pten, pik3ca, met, top2a, and mdm2 - Google Patents

Abstract

Disclosed herein are nucleic acid probes comprising a plurality of segments of uniquely specific nucleic acid sequences. In some examples, the nucleic acid probes include or consist of a nucleic acid sequence set forth as any one of SEQ ID NOs: 1-50. The disclosed probes are useful for detecting presence of a target nucleic acid in a sample. In some embodiments, the probes are useful for detecting presence of phosphatase and tensin homolog (PTEN), phosphatidylinositol 3-kinase p110 (PIK3CA), mouse double minute 2 (MDM2), Met proto-oncogene (MET), or topoisomerase II alpha (TOP2A) in a sample.

Description

UNIQUELY SPECIFIC PROBES FOR PTEN, PIK3CA, MET, TOP2A,

AND MDM2

FIELD

This disclosure relates to detection of nucleic acid target sequences (e.g., genomic DNA or RNA), particularly probes for detection of a target nucleic acid, and methods of their use. In some embodiments, the disclosed probes include uniquely specific nucleic acid sequences.

BACKGROUND

Molecular cytogenetics methods are based on hybridization of a nucleic acid probe to its complementary nucleic acid within a cell. A probe for a specific chromosomal region will recognize and hybridize to its complementary sequence on a metaphase chromosome or within an interphase nucleus (for example in a tissue sample). Probes have been developed for a variety of diagnostic and research purposes. Hybridization of chromosome or gene-specific probes has made possible detection of chromosomal abnormalities associated with numerous diseases and syndromes, including constitutive genetic anomalies, such as microdeletion syndromes, chromosome translocations, gene amplification and aneuploidy syndromes, neoplastic diseases, as well as pathogen infections. Most commonly these techniques are applied to standard cytogenetic preparations on microscope slides. In addition, these procedures can be used on slides of formalin- fixed tissue, blood or bone marrow smears, and directly fixed cells or other nuclear isolates.

Many cancers are characterized genetic changes that lead to aberrant control of cellular processes, or to uncontrolled growth and proliferation of cells. These genetic changes include gain or loss of function (for example, including amplification or deletion of all or a portion of a gene), gene rearrangement, and changes in sequence (for example, substitution, addition, or deletion or one or more bases). Such changes are known to occur in the genetic regions associated with various genes, including phosphatase and tensin homolog (PTEN), phosphatidylinositol 3-kinase pi 10 (PIK3CA), mouse double minute 2 (MDM2), Met proto-oncogene (MET), and topoisomerase II alpha (TOP2A) genes. In addition to their well-known applicability to genetic abnormalities associated with cancer, abnormalities in these regions have been associated with autism spectrum disorders, metabolism, motoneuron specification, and cardiovascular disease. Detection of genetic changes in these regions can provide diagnostic and prognostic information for patients and in some cases, inform treatment decisions.

SUMMARY

Disclosed herein are nucleic acid probes that include a plurality of segments of uniquely specific nucleic acid sequences. The disclosed probes are useful for detecting presence of a target nucleic acid in a sample. The uniquely specific nucleic acid sequences are designed to occur only once each in the haploid genome of an organism, and provide high levels of sensitivity and specificity for the detection of a target nucleic acid in a sample. In some embodiments, the probes are useful for detecting presence and location of PTEN, PIK3CA, MDM2, MET, or TOP2A nucleic acids in a sample.

In some embodiments, the disclosed probes include isolated nucleic acid molecules comprising the sequences provided herein as any one of SEQ ID NOs: 1-50, or nucleic acids having at least 70% (such as at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity with any one of SEQ ID NOs: 1-50. In other embodiments, the disclosed probes include isolated nucleic acid molecules comprising at least 70% (such as at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity with at least 250 contiguous nucleotides of any one of SEQ ID NOs: 1-50. In some examples, the nucleic acid probes include nucleic acid molecules consisting of the sequence of any one of SEQ ID NOs: 1-50 or at least 250 contiguous nucleotides of any one of SEQ ID NOs: 1-50. In some examples, the disclosed probes include a detectable label.

Also disclosed herein are probe sets for detecting a target nucleic acid molecule. A probe set includes two or more (for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) of the disclosed nucleic acid probes. In some examples a probe set useful for detecting PTEN includes two or more nucleic acid molecules selected from SEQ ID NOs: 1-10. In other example, a probe set useful for detecting PIK3CA includes two or more nucleic acid molecules selected from SEQ ID NOs: 1 1-20. In another example, a probe set useful for detecting MDM2 includes two or more nucleic acid molecules selected from SEQ ID NOs: 21-30. In a still further example, a probe set useful for detecting MET includes two or more nucleic acid molecules selected from SEQ ID NOs: 31-40. In another example, a probe set useful for detecting TOP2A includes two or more nucleic acid molecules selected from SEQ ID NOs: 41-50. Also disclosed are kits including one or more of the probes or probe sets disclosed herein.

Methods of using the disclosed probes include, for example, detecting (and in some examples quantifying) a target nucleic acid. For example, the method can include contacting one or more of the disclosed probes or probe sets with a sample containing nucleic acid molecules under conditions sufficient to permit hybridization between the nucleic acid molecules in the sample and the one or more probes. Resulting hybridization is detected, wherein the presence of hybridization indicates the presence (and in some examples, the quantity) of the target nucleic acid sequence. In some embodiments, methods of using the disclosed probes include detecting copy number of the target nucleic acid.

The foregoing and other features of the disclosure will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.

FIG. 1 is a digital image of dual in situ hybridization (ISH) of breast tissue using the PTEN probe set SEQ ID NOs: 1-10 and a chromosome 10 centromere probe.

FIG. 2 is a digital image of dual ISH of breast tissue using the PIK3CA probe set SEQ ID NOs: 1 1-20 and a chromosome 3 centromere probe.

FIG. 3 is a digital image of dual ISH of liposarcoma using the MDM2 probe set SEQ ID NOs: 21-30 and a chromosome 12 centromere probe.

FIG. 4 is a digital image of dual ISH of lung tissue using the MET probe set SEQ ID NOs: 31-40 and a chromosome 7 centromere probe.

FIG. 5 is a digital image of dual ISH of breast tissue using the TOP2A probe set SEQ ID NOs: 41-50 and a chromosome 17 centromere probe.

SEQUENCES

The nucleic acid sequences provided herein are shown using standard letter abbreviations for nucleotide bases, as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand. In the provided sequences: SEQ ID NOs: 1-10 are nucleic acid sequences of probes including linked uniquely specific nucleic acid segments complementary to the human PTEN gene.

SEQ ID NOs: 1 1-20 are nucleic acid sequences of probes including linked uniquely specific nucleic acid segments complementary to the human PIK3CA gene.

SEQ ID NOs: 21-30 are nucleic acid sequences of probes including linked uniquely specific nucleic acid segments complementary to the human MDM2gene.

SEQ ID NOs: 31-40 are nucleic acid sequences of probes including linked uniquely specific nucleic acid segments complementary to the human MET gene.

SEQ ID NOs: 41-50 are nucleic acid sequences of probes including linked uniquely specific nucleic acid segments complementary to the human TOP2A gene.

DETAILED DESCRIPTION

I. Abbreviations

aCGH array comparative genomic hybridization

AP alkaline phosphatase

CGH comparative genomic hybridization

CISH chromogenic in situ hybridization

DNP 2,4-dinitrophenyl

FISH fluorescent in situ hybridization

HRP horseradish peroxidase

ISH in situ hybridization

MDM2 murine double minute 2

MET Met proto-oncogene

PIK3CA phosphatidylinositol 3-kinase (p 110)

PTEN phosphatase and tension homolog

SISH silver in situ hybridization

TOP2A topoisomerase II alpha

II. Terms

Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which a disclosed invention belongs. The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. "Comprising" means "including." Hence "comprising A or B" means "including A" or "including B" or "including A and B."

Suitable methods and materials for the practice and/or testing of embodiments of the disclosure are described below. Such methods and materials are illustrative only and are not intended to be limiting. Other methods and materials similar or equivalent to those described herein can be used. For example, conventional methods well known in the art to which the disclosure pertains are described in various general and more specific references, including, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, 1989; Sambrook et al., Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Press, 2001 ; Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates, 1992 (and Supplements to 2000); Ausubel et al., Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, 4th ed., Wiley & Sons, 1999; Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1990; and Harlow and Lane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor

Laboratory Press, 1999.

All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety for all purposes. In case of conflict, the present specification, including explanations of terms, will control.

Although methods and materials similar or equivalent to those described herein can be used to practice or test the disclosed technology, suitable methods and materials are described below. The materials, methods, and examples are illustrative only and not intended to be limiting.

In order to facilitate review of the various embodiments of the disclosure, the following explanations of specific terms are provided:

Detectable label: A compound or composition that is conjugated directly or indirectly to another molecule (such as a nucleic acid probe) to facilitate detection of that molecule. Specific, non- limiting examples of labels include fluorescent and fluorogenic moieties, chromogenic moieties, haptens, affinity tags, and radioactive isotopes. The label can be directly detectable (e.g., optically detectable) or indirectly detectable (for example, via interaction with one or more additional molecules that are in turn detectable).

Exemplary labels in the context of the probes disclosed herein are described below.

Methods for labeling nucleic acids, and guidance in the choice of labels useful for various purposes, are discussed, e.g., in Sambrook and Russell, in Molecular Cloning: A

Laboratory Manual, 3^rd Ed., Cold Spring Harbor Laboratory Press (2001) and Ausubel et al, in Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley- Intersciences (1987, and including updates).

Hybridization: To form base pairs between complementary regions of two strands of DNA, RNA, or between DNA and RNA, thereby forming a duplex molecule. Hybridization conditions resulting in particular degrees of stringency will vary depending upon the nature of the hybridization method and the composition and length of the hybridizing nucleic acid sequences. Generally, the temperature of hybridization and the ionic strength (such as the Na⁺ concentration) of the hybridization buffer will determine the stringency of hybridization. The presence of a chemical which decreases hybridization (such as formamide) in the hybridization buffer will also determine the stringency (Sadhu et al, J. Biosci. 6:817-821, 1984). Calculations regarding hybridization conditions for attaining particular degrees of stringency are discussed in Sambrook et al., (1989) Molecular Cloning, second edition, Cold Spring Harbor Laboratory, Plainview, NY (chapters 9 and 1 1). Hybridization conditions for ISH are also discussed in Landegent et al., Hum. Genet. 77:366-370, 1987; Lichter et al, Hum. Genet. 80:224-234, 1988; and Pinkel et al, Proc. Natl. Acad. Sci. USA 85:9138-9142, 1988.

Intron: An intron is any nucleotide region or sequence within a gene that is removed by RNA splicing during generation of a final mature RNA product of a gene. The term intron may refer to both the DNA sequence within a gene or the corresponding sequence in unprocessed RNA transcripts. Introns are found in the genes of most organisms, and can be located in a wide range of genes, including those that generate proteins, ribosomal RNA (rRNA), and transfer RNA (tRNA). When proteins are generated from intron-containing genes, RNA splicing takes place as part of the RNA processing pathway that follows transcription and precedes translation. The length of intron sequences is highly variable, ranging from less than 100 base pairs to tens of thousands or even hundreds of thousands of base pairs.

Isolated: An "isolated" biological component (such as a nucleic acid molecule, protein, or cell) has been substantially separated or purified away from other biological components in a preparation, a cell of an organism, or the organism itself, in which the component occurs, such as other chromosomal and extra-chromosomal DNA and RNA, proteins and cells. Nucleic acid molecules and proteins that have been "isolated" include nucleic acid molecules and proteins purified by standard purification methods. The term also embraces nucleic acid molecules and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acid molecules and proteins. In some examples, the nucleic acid probes disclosed herein are isolated nucleic acid probes. Met proto-oncogene (MET): Also known as c-Met, HGFR, AUTS9, RCCP2, or MN G HOS Transforming gene. MET is a proto-oncogene that encodes a protein known as hepatocyte growth factor receptor (HGFR). The human MET proto-oncogene (GenelD: 4233) usually has a total length of 125,982 bp, and it is located in the 7q31 locus of chromosome 7. Human MET is transcribed into a 6,641 bp mature mRNA, which is then translated into a 1,390 amino-acid MET protein. The sequence of the human MET proto- oncogene was disclosed as early as 1987 by Park et al. (Proc. Natl. Acad. Sci. USA 84:6379-6383, 1987).

Mouse double minute 2 (MDM2): The human gene is also known as p53 E3 ubiquitin protein ligase homolog (mouse). The MDM2 gene was originally identified by virtue of its amplification in a spontaneously transformed derivative of mouse BALB/c cells, and the MDM2 protein was subsequently shown to bind to p53 in rat cells transfected with p53 genes. Momand et al. (Cell 69: 1237-1245, 1992) characterized, purified, and identified a cellular phosphoprotein with an apparent molecular mass of 90 kD that formed a complex with both mutant and wild-type p53 protein. The protein was identified as a product of the murine double minute 2 gene (mdm2). Oliner et al. (Nature 358:80-83, 1992) cloned the human MDM2 gene. By fluorescence in situ hybridization onto simultaneously DAPI -banded metaphase chromosomes and interphase nuclei, Mitchell et al. (Chromosome Res. 3:261-262, 1995) mapped MDM2 to human 12ql4.3- ql5 distal to CDK4 and flanked by Genethon microsatellites D12S80 and D12S83.

Momand et al. found that the MDM2 gene enhances the tumorigenic potential of cells when it is overexpressed and encodes a putative transcription factor. Forming a tight complex with the p53 gene, the MDM2 oncogene can inhibit p53-mediated transactivation. Because the 12ql3-ql4 region, to which the MDM2 gene maps, is often aberrant in human sarcomas, Oliner et al. performed Southern blot analysis on DNA from such cancers. A striking amplification of MDM2 sequences was found in 17 of 47 sarcomas, including common bone and soft tissue forms. The results were considered consistent with the hypothesis that MDM2 binds to p53 and that amplification of MDM2 in sarcomas leads to escape from p53-regulated growth control. This mechanism of tumorigenesis parallels that for virus-induced tumors in which viral oncogene products bind to and functionally inactivate p53.

Phosphatase and tensin homolog (PTEN): Also referred to as MMAC1 (mutated in multiple advanced cancers). A gene that, in humans, encodes a ubiquitously expressed tumor suppressor dual-specificity phosphatase that antagonizes the

phosphatidylinositol 3 -kinase signaling pathway through its lipid phosphatase activity and negatively regulates the MAP kinase pathway through its protein phosphatase activity (Pezzolesi et al, Hum. Mol. Genet. 16: 1058-1071, 2007). PTEN acts as a tumor suppressor gene through the action of its phosphatase protein product. This phosphatase is involved in the regulation of the cell cycle, preventing cells from growing and dividing too rapidly. Mutations of this gene are a step in the development of many cancers. The sequence of human PTEN was disclosed as early as 1997 by Li et al. (J. Biol. Chem. 272:29403-29406, 1997).

Phosphatidylinositol 3-kinase, pllO subunit (PIK3CA): Also known as phosphoinositide-3 -kinase, catalytic, alpha polypeptide. Human phosphatidylinositol 3- kinase (EC 2.7.1.137) is composed of 85-kD and 1 10-kD subunits. The 85-kD subunit lacks phosphatidylinositol 3-kinase activity and acts as an adaptor, coupling the 1 10-kD subunit (pi 10) to activated protein tyrosine kinases. The human pi 10 subunit is referred to herein as PIK3CA. Hiles et al. (Cell 70:419-429, 1992) found that the human cDNA for pi 10 predicts a 1,068-amino acid protein related to a protein which in S. cerevisiae is involved in the sorting of proteins to the vacuole. In COS-1 cells, pi 10 was catalytically active only when complexed with p85-alpha. Volinia et al. (Genomics 24:472-477^', 1994) contributed to the structural and functional understanding of phosphatidylinositol 3-kinase by purifying, cloning, and subsequently elucidating the expression of the bovine enzyme. cDNA for the human PIK3CA encodes a protein 99% identical to the bovine pi 10. The chromosomal localization of the gene for human PIK3CA is shown to be at 3q21-qter as determined using somatic cell hybrids. In situ hybridization performed using Alu-PCR from the YAC DNA located the human gene in 3q26.3 [Chromosome 3: 178,865,902- 178,957,881]. The sequence for the human PIK3CA gene was disclosed as early as 1994 by Volinia et al.

Probe: A nucleic acid molecule that is capable of hybridizing with a target nucleic acid molecule (e.g., genomic target nucleic acid molecule) and, when hybridized to the target, is capable of being detected either directly or indirectly. Thus probes permit the detection, and in some examples quantification, of a target nucleic acid molecule. In particular examples, a probe includes at least two segments complementary to uniquely specific nucleic acid sequences of a target nucleic acid molecule and are thus capable of specifically hybridizing to at least a portion of the target nucleic acid molecule. Generally, once at least one segment or portion of a segment has (and remains) hybridized to the target nucleic acid molecule other portions of the probe may (but need not) be physically constrained from hybridizing to those other portions' cognate binding sites in the target (e.g., such other portions are too far distant from their cognate binding sites); however, other nucleic acid molecules present in the probe can bind to one another, thus amplifying signal from the probe. A probe can be referred to as a "labeled nucleic acid probe," indicating that the probe is coupled directly or indirectly to a detectable moiety or "label," which renders the probe detectable.

Sample: A specimen containing DNA (for example, genomic DNA), RNA

(including mRNA), protein, or combinations thereof, obtained from a subject. Examples include, but are not limited to, chromosomal preparations, peripheral blood, urine, saliva, tissue biopsy, fine needle aspirate, surgical specimen, bone marrow, amniocentesis samples, and autopsy material. In one example, a sample includes genomic DNA. In some examples, the sample is a cytogenetic preparation, for example which can be placed on microscope slides. In particular examples, samples are used directly, or can be manipulated prior to use, for example, by fixing (e.g., using formalin).

Sequence identity: The identity (or similarity) between two or more nucleic acid sequences is expressed in terms of the identity or similarity between the sequences.

Sequence identity can be measured in terms of percentage identity; the higher the percentage, the more identical the sequences are. Sequence similarity can be measured in terms of percentage similarity (which takes into account conservative amino acid substitutions); the higher the percentage, the more similar the sequences are.

Methods of alignment of sequences for comparison are well known in the art.

Various programs and alignment algorithms are described in: Smith & Waterman, Adv. Appl. Math. 2:482, 1981 ; Needleman & Wunsch, J. Mol. Biol. 48:443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988; Higgins & Sharp, Gene, 73:237-44, 1988; Higgins & Sharp, CABIOS 5: 151-3, 1989; Corpet et al, Nuc. Acids Res. 16: 10881-90, 1988; Huang et al. Computer Appls. in the Biosciences 8: 155-65, 1992; and Pearson et al., Meth. Mol. Bio. 24:307-31, 1994. Altschul et al, J. Mol. Biol. 215:403-10, 1990, presents a detailed consideration of sequence alignment methods and homology calculations.

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al, J. Mol. Biol. 215:403-10, 1990) is available from several sources, including the National Center for Biotechnology and on the Internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. Additional information can be found at the NCBI web site.

BLASTN may be used to compare nucleic acid sequences, while BLASTP may be used to compare amino acid sequences. If the two compared sequences share homology, then the designated output file will present those regions of homology as aligned sequences. If the two compared sequences do not share homology, then the designated output file will not present aligned sequences.

The BLAST-like alignment tool (BLAT) may also be used to compare nucleic acid sequences (Kent, Genome Res. 12:656-664, 2002). BLAT is available from several sources, including Kent Informatics (Santa Cruz, CA) and on the Internet

(genome.ucsc.edu).

Once aligned, the number of matches is determined by counting the number of positions where an identical nucleotide or amino acid residue is presented in both sequences. The percent sequence identity is determined by dividing the number of matches either by the length of the sequence set forth in the identified sequence, or by an articulated length (such as 100 consecutive nucleotides or amino acid residues from a sequence set forth in an identified sequence), followed by multiplying the resulting value by 100. For example, a nucleic acid sequence that has 1 166 matches when aligned with a test sequence having 1554 nucleotides is 75.0 percent identical to the test sequence (1 166÷1554* 100=75.0). The percent sequence identity value is rounded to the nearest tenth. For example, 75.1 1, 75.12, 75.13, and 75.14 are rounded down to 75.1, while 75.15, 75.16, 75.17, 75.18, and 75.19 are rounded up to 75.2. The length value will always be an integer. In another example, a target sequence containing a 20-nucleotide region that aligns with 15 consecutive nucleotides from an identified sequence as follows contains a region that shares 75 percent sequence identity to that identified sequence (that is, 15÷20* 100=75).

Subject: Any multi-cellular vertebrate organism, such as human or non-human mammals (e.g., veterinary subjects).

Target nucleic acid sequence or molecule: A defined region or particular portion of a nucleic acid molecule, for example a portion of a genome (such as a gene or a region of mammalian genomic DNA containing a gene of interest). In an example where the target nucleic acid sequence is a target genomic sequence, such a target can be defined by its position on a chromosome (e.g., in a normal cell), for example, according to cytogenetic nomenclature by reference to a particular location on a chromosome; by reference to its location on a genetic map; by reference to a hypothetical or assembled contig; by its specific sequence or function; by its gene or protein name; or by any other means that uniquely identifies it from among other genetic sequences of a genome. In some examples, the target nucleic acid sequence is mammalian genomic sequence (for example human genomic sequence). In some examples, alterations of a target nucleic acid sequence (e.g., genomic nucleic acid sequence) are "associated with" a disease or condition. In some examples, detection of the target nucleic acid sequence can be used to infer the status of a sample with respect to the disease or condition. For example, the target nucleic acid sequence can exist in two (or more) distinguishable forms, such that a first form correlates with absence of a disease or condition and a second (or different) form correlates with the presence of the disease or condition. The two different forms can be qualitatively distinguishable, such as by polynucleotide polymorphisms, and/or the two different forms can be quantitatively distinguishable, such as by the number of copies of the target nucleic acid sequence that are present in a cell.

Topoisomerase II alpha (TOP2A): DNA topoisomerases (EC 5.99.1.3) are enzymes that control and alter the topologic states of DNA in both prokaryotes and eukaryotes. Topoisomerase II from eukaryotic cells catalyzes the relaxation of supercoiled DNA molecules, catenation, decatenation, knotting, and unknotting of circular DNA. The reaction catalyzed by topoisomerase II likely involves the crossing-over of two DNA segments. The gene encoding topoisomerase II in humans is present at cytogenetic location: 17q21.2 and has genomic coordinates (GRCh37): 17:38,544,772 - 38,574,201. Tsai-Pflugfelder et al. determined the entire coding sequence of the human TOP2 gene as early as 1988 (Proc. Natl. Acad. Sci. USA 85:7177-7181, 1988). Lang et al. reported the complete structures of the human TOP2A and TOP2B genes in 1998 (Gene 221 :255-266, 1998). The human TOP2A gene spans approximately 30 kb and contains 35 exons.

Uniquely specific sequence: A nucleic acid sequence (for example, a sequence of at least of at least 20 bp (such as at least 20 bp, 30 bp, 40 bp, 50 bp, 60 bp, 70 bp, 80 bp, 90 bp, 100 bp, or more) that is present only one time in a haploid genome of an organism. In a particular example, a uniquely specific nucleic acid sequence is a nucleic acid sequence from a target nucleic acid that has 100% sequence identity with the target nucleic acid and has no significant identity to any other nucleic acid sequences present in the specific haploid genome that includes the target nucleic acid.

Vector: Any nucleic acid that acts as a carrier for other ("foreign") nucleic acid sequences that are not native to the vector. When introduced into an appropriate host cell a vector may replicate itself (and, thereby, the foreign nucleic acid sequence) or express at least a portion of the foreign nucleic acid sequence. In one context, a vector is a linear or circular nucleic acid into which a nucleic acid sequence of interest is introduced (for example, cloned) for the purpose of replication (e.g., production) and/or manipulation using standard recombinant nucleic acid techniques (e.g., restriction digestion). A vector can include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication. A vector can also include one or more selectable marker genes and other genetic elements known in the art. Common vectors include, for example, plasmids, cosmids, phage, phagemids, artificial chromosomes (e.g., BAC, PAC, HAC, YAC), and hybrids that incorporate features of more than one of these types of vectors. Typically, a vector includes one or more unique restriction sites (and in some cases a multi-cloning site) to facilitate insertion of a target nucleic acid sequence.

III. Uniquely Specific Nucleic Acid Probes

Disclosed herein are uniquely specific nucleic acid probes complementary to target nucleic acid molecules of interest, including PTEN, PIK3CA, MDM2, MET and TOP2A. Each of the disclosed probes includes a plurality of uniquely specific nucleic acid segments, which were designed and synthesized utilizing the methods described in U.S. Pat. App. Publ. No. 201 1/0160076 and International Pat. Publ. No. WO 201 1/062293, both of which are incorporated herein by reference in their entirety. The nucleic acid segments included in each probe are each uniquely specific (occur only once in the haploid human genome) and are from non-contiguous portions of the human genome.

Surprisingly, many of the segments are located in introns in the human genome. While not being bound by theory, it is generally believed in the field that the intronic regions of the human genome evolve at a faster rate than coding regions. In illustrative embodiments, the present probes have sequences that are exon-free or substantially exon- free. In one embodiment, the sequences are selected from non-coding gene regions. The development and use of intronic probes is counter-intuitive as most of the field is concerned with genetic abnormalities associated with the protein coding region of the gene. The present probes provide researchers and clinicians with unique and distinct data heretofore unavailable. It is unexpected that introns include uniquely specific sequences due to the belief in the field that introns are not as highly conserved as the exon-containing DNA sequences. The identification of uniquely specific sequences in intronic regions indicates that some of these regions may be highly conserved. The present probes provide a means to study these highly-conserved intronic regions of these very important genes.

A. PTEN

Disclosed herein are isolated nucleic acid probes including linked uniquely specific nucleic acid segments complementary to the human PTEN gene (e.g., Gene ID No. 5728; NC_000010.10 (89623195..89728532), incorporated herein by reference as present in GENBANK® on April 30, 2012). In some embodiments, the nucleic acid probes include or consist of nucleic acid molecules with at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or more sequence identity to the nucleotide sequences set forth in SEQ ID NOS: 1- 10.

In some embodiments, the nucleic acid probe can have a sequence with at least

70%, at least 75%, at least 80%, at least 85%, or at least 90% sequence identity (for example at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to one of the nucleotide sequences set forth in SEQ ID NOs: 1-10. In other embodiments, the nucleic acid probe can include a sequence with at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% sequence identity (for example at least 91%, 92%, 93%, 94%,

95%, 96%, 97%, 98%, 99%, or 100% identity) to at least 250 contiguous nucleotides (such as at least 400, at least 500, at least 750, at least 1000, at least 2000, at least 3000, at least 4000, or at least 5000 contiguous nucleotides) of any one of SEQ ID NOs: 1-10. In some examples, the nucleic acid probes include or consist of the sequences set forth in SEQ ID NOs: 1-10. In one example, the probe includes or consists of a contiguous nucleic acid molecule comprising two or more of SEQ ID Nos. 1-10 (such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 of SEQ ID NOs: 1-10) in a single nucleic acid molecule.

B. PIK3CA

Disclosed herein are isolated nucleic acid probes including linked uniquely specific nucleic acid segments complementary to the human PIK3CA gene {e.g., Gene ID No. 5290; NC_000003.1 1 (17886631 1..178952500), incorporated herein by reference as present in GENBANK® on April 30, 2012). In some embodiments, the isolated nucleic acid probes include or consist of nucleic acid molecules with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or more sequence identity to the nucleotide sequences set forth in SEQ ID NOs. 1 1-20.

In some embodiments, the nucleic acid probe can have a sequence with at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% sequence identity (for example at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to one of the nucleotide sequences set forth in SEQ ID NOs: 1 1-20. In other embodiments, the nucleic acid probe can include a sequence with at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% sequence identity (for example at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to at least 250 contiguous nucleotides (such as at least 400, at least 500, at least 750, at least 1000, at least 2000, at least 3000, at least 4000, or at least 5000 contiguous nucleotides) of any one of SEQ ID NOs: 1 1-20. In some examples, the nucleic acid probes include or consist of the sequences set forth in SEQ ID NOs: 1 1-20. In one example, the probe includes or consists of a contiguous nucleic acid molecule comprising two or more of SEQ ID NOs: 1 1-20 (such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 of SEQ ID NOs: 1 1-20) in a single nucleic acid molecule.

C. MDM2

Disclosed herein are isolated nucleic acid probes including linked uniquely specific nucleic acid segments complementary to the human MDM2 gene (e.g., Gene ID No. 4193; NC 000012.1 1 (69201971..69239212), incorporated herein by reference as present in GENBANK® on April 30, 2012). In some embodiments, the isolated nucleic acid probes include or consist of nucleic acid molecules with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or more sequence identity to the nucleotide sequences set forth in SEQ ID NOs. 21-30.

In some embodiments, the nucleic acid probe can have a sequence with at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% sequence identity (for example at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to one of the nucleotide sequences set forth in SEQ ID NOs: 21-30. In other embodiments, the nucleic acid probe can include a sequence with at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% sequence identity (for example at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to at least 250 contiguous nucleotides (such as at least 400, at least 500, at least 750, at least 1000, at least 2000, at least 3000, at least 4000, or at least 5000 contiguous nucleotides) of any one of SEQ ID NOs: 21-30. In some examples, the nucleic acid probes include or consist of the sequences set forth in SEQ ID NOs: 21-30. In one example, the probe includes or consists of a contiguous nucleic acid molecule comprising two or more of SEQ ID NOs: 21-30 (such as 2, 3, 4, 5, 6, 7, 8, 9, or

10 of SEQ ID NOs: 21-30) in a single nucleic acid molecule.

D. MET

Disclosed herein are isolated nucleic acid probes including linked uniquely specific nucleic acid segments complementary to the human MET gene (e.g., Gene ID No.

4233; NC 000007.13 (1 16312459..1 16438440), incorporated herein by reference as present in GENBANK® on April 30, 2012). In some embodiments, the isolated nucleic acid probes include or consist of nucleic acid molecules with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or more sequence identity to the nucleotide sequence set forth in SEQ ID NOs. 31-40. In some embodiments, the nucleic acid probe can have a sequence with at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% sequence identity (for example at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to one of the nucleotide sequences set forth in SEQ ID NOs: 31-40. In other embodiments, the nucleic acid probe can include a sequence with at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% sequence identity (for example at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to at least 250 contiguous nucleotides (such as at least 400, at least 500, at least 750, at least 1000, at least 2000, at least 3000, at least 4000, or at least 5000 contiguous nucleotides) of any one of SEQ ID NOs: 31-40. In some examples, the nucleic acid probes include or consist of the sequences set forth in SEQ ID

NOs: 31-40. In one example, the probe includes or consists of a contiguous nucleic acid molecule comprising two or more of SEQ ID NOs: 31-40 (such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 of SEQ ID NOs: 31-40) in a single nucleic acid molecule. E. TOP 2 A

Disclosed herein are isolated nucleic acid probes including linked uniquely specific nucleic acid segments complementary to the human TOP2A gene {e.g., Gene ID No.7153; NC_000017.10 (38544773..38574202, complement), incorporated herein by reference as present in GENBANK® on April 30, 2012). In some embodiments, the isolated nucleic acid probes include or consist of nucleic acid molecules with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or more sequence identity to the nucleotide sequences set forth in SEQ ID NOs. 41-50.

In some embodiments, the nucleic acid probe can have a sequence with at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% sequence identity (for example at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to one of the nucleotide sequences set forth in SEQ ID NOs: 41-50. In other embodiments, the nucleic acid probe can include a sequence with at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% sequence identity (for example at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to at least 250 contiguous nucleotides (such as at least 400, at least 500, at least 750, at least 1000, at least 2000, at least 3000, at least

4000, or at least 5000 contiguous nucleotides) of any one of SEQ ID NOs: 41-50. In some examples, the nucleic acid probes include or consist of the sequences set forth in SEQ ID NOs: 41-50. In one example, the probe includes or consists of a contiguous nucleic acid molecule comprising two or more of SEQ ID NOs: 41-50 (such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 of SEQ ID NOs: 41-50) in a single nucleic acid molecule. In some embodiments, the disclosed probes have a length of at least 250 contiguous nucleotides (such as at least 300, at least 400, at least 500, at least 750, at least 1000, at least 2000, at least 3000, at least 4000, at least 5000, at least 6000, at least 7000, at least 8000, at least 9000, at least 10,000, at least 12,000, at least 15,000, at least 20,000, at least 25,000, at least 30,000, at least 35,000, at least 40,000, at least 45,000, at least

50,000, or more contiguous nucleotides). In other embodiments, the disclosed probes have a length of about 250-50,000 nucleotides (for example, about 500-50,000, about 1000- 40,000, about 5000-25,000, about 7000-20,000, or about 10,000 to 15,000 nucleotides). The probes can include all or a portion of one or more of SEQ ID NOs: 1-50, or all or a portion of a nucleic acid having at least 70% sequence identity (for example, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity) to one of more of SEQ ID NOs: 1-50. In some examples, the probe is a contiguous nucleic acid molecule comprising up to 10 of the disclosed sequences (such as SEQ ID NOs: 1- 10, SEQ ID NOs: 1 1-20, SEQ ID NOs: 21-30, SEQ ID NOs: 31-40, or SEQ ID NOs: 41-50).

In other examples, the probe is a contiguous nucleic acid molecule including two or more portions or segments, wherein the first portion includes at least 250 contiguous nucleotides at least 70% identical (for example, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the corresponding portion of any one of SEQ ID NOs: 1-50 and the second portion includes at least 250 contiguous nucleotides at least 70% identical (for example, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the corresponding portion of any one of SEQ ID NOs: 1-50, where the first and second portions are different.

Each of the disclosed sequences of SEQ ID NOs: 1-50 include a plurality of 100 bp uniquely specific nucleic acid segments, which were designed and synthesized utilizing the methods described in U.S. Pat. App. Publ. No. 201 1/0160076 and International Pat. Publ. No. WO 201 1/062293, both of which are incorporated herein by reference in their entirety. The disclosed probes are exemplary, and it is understood that in some examples, at least one (such as at least two, at least three, at least four, at least five, or more) of the 100 bp segments (for example nucleotides 1-100, 101-200, 201-300, and so on) or portions thereof could be removed from the disclosed sequences to provide additional nucleic acid probes for PTEN, PIK3CA, MDM2, MET, or TOP2A. In other examples, the order of at least one (such as at least two, at least three, at least four, at least five, or more) of the 100 bp segments (for example nucleotides 1-100, 101-200, 201-300, and so on) could be rearranged from the order in the disclosed probes to provide additional nucleic acid probes for PTEN, PIK3CA, MDM2, MET, or TOP2A. One of skill in the art could make and test such modified probes, for example by synthesizing a modified probe and assessing its hybridization to samples known to contain or not to contain the target nucleic acid.

In some examples, the individual uniquely specific segments are produced (for example by oligonucleotide synthesis or by amplification of the sequences from the genomic target nucleic acid) and joined together. In other examples, the nucleic acid probe is synthesized as a series of oligonucleotides (such as individual oligonucleotides of about 100 bp), which are joined together. For example, the segments may be joined or ligated to one another enzymatically (e.g., using a ligase). For example, the segments can be joined in a blunt-end ligation or at a restriction site. In another example, the segments may be synthesized with complementary nucleic acid overhangs (such as at least a 3 bp overhang), annealed, and joined to one another, for example with a ligase. Chemical ligation and amplification can also be used to join the uniquely specific segments. In another example, the entire nucleic acid probe is synthesized and the individual uniquely specific segments are directly joined during synthesis.

In some embodiments, the disclosed nucleic acid probes are included in a vector, such as a plasmid or an artificial chromosome (e.g., yeast artificial chromosome (YAC), PI based artificial chromosome (PAC), or bacterial artificial chromosome (BAC)). In some examples, any one of SEQ ID NOs: 1-50 disclosed herein are introduced into a plasmid vector, for example to allow replication and/or labeling of the nucleic acid probe by standard molecular biology techniques. In one example, the vector is a pUC plasmid vector. In other examples, two or more of the disclosed sequences (such as 2, 3, 4, 5, 6, 7, 8, 9, 10, or more of the disclosed sequences) are introduced into a vector, for example to allow replication and/or labeling of the nucleic acid probe by standard molecular biology techniques. The two or more sequences can be introduced into a vector in any order. One of skill in the art can determine whether the sequences the overlap the junctions between the individual sequences (for example, a window of about 100 bp) are uniquely specific, for example, utilizing the teachings of U.S. Pat. App. Publ. No. 201 1/0160076 and

International Pat. Publ. No. WO 2011/062293. If a sequence that is not uniquely specific is introduced, the sequences can be reordered and reanalyzed in order to select an order that does not produce any non-uniquely specific sequences. In particular examples, all of SEQ ID NOs: 1-10 are introduced into a single vector, all of SEQ ID NOs: 1 1-20 are introduced into a single vector, all of SEQ ID NOs: 21-30 are introduced into a single vector, all of SEQ ID NOs: 31-40 are introduced into a single vector, or all of SEQ ID NOs: 41-50 are introduced into a single vector. In one example, the vector is an artificial chromosome (such as a YAC, BAC or PAC).

IV. Probe Sets

In some embodiments, the disclosed probes are included in a set of probes, for example as set of two or more (such as 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) of the disclosed probes. In some examples, a probe set includes two or more probes specific for a particular target nucleic acid molecule (such as two or more probes for PTEN, PIK3CA, MET, MDM2, or TOP2A). In some examples, a probe set includes probes specific for two or more target nucleic acid molecules (such as probes specific for two or more of PTEN, PIK3CA, MET, MDM2, and TOP2A).

One exemplary probe set includes two or more probes specific for human PTEN. The probe set includes two or more (such as 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) probes selected from probes including or consisting of the nucleic acid sequence having at least at least 70%, at least 75%, at least 80%, at least 85%, or 90% sequence identity with SEQ ID NOs: 1-10 or at least 250 contiguous nucleotides of any one of SEQ ID NOs: 1-10. In one example, a probe set for PTEN includes probes including or consisting of nucleic acid sequences having at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% sequence identity with each of SEQ ID NOs: 1-10. In a specific example, the probe set includes each of SEQ ID NOs: 1-10.

Another exemplary probe set includes probes specific for human PIK3CA. The probe set includes two or more (such as 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) probes selected from probes including or consisting of the nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% sequence identity with any one of SEQ ID NOs: 1 1-20 or at least 250 contiguous nucleotides of any one of SEQ ID NOs: 1 1- 20. In one example, a probe set for PIK3CA includes probes including or consisting of nucleic acid sequences having at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% sequence identity with each of SEQ ID NOs: 1 1-20. In a particular example, the probe set includes each of SEQ ID NOs: 1 1-20.

A further exemplary probe set includes probes specific for human MDM2. The probe set includes two or more (such as 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) probes selected from probes including or consisting of the nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% sequence identity with any one of SEQ ID NOs: 21-30 or at least 250 contiguous nucleotides of any one of SEQ ID NOs: 21- 30. In one example, a probe set for MDM2 includes probes including or consisting of nucleic acid sequences having at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% sequence identity with each of SEQ ID NOs: 21-30. In a particular example, the probe set includes each of SEQ ID NOs: 21-30.

A still further exemplary probe set includes probes specific for human MET. The probe set includes two or more (such as 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) probes selected from probes including or consisting of the nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% sequence identity with any one of SEQ ID NOs: 31-40 or at least 250 contiguous nucleotides of any one of SEQ ID NOs: 31- 40. In one example, a probe set for MET includes probes including or consisting of nucleic acid sequences having at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% sequence identity with each of SEQ ID NOs: 31-40. In a particular example, the probe set includes each of SEQ ID NOs: 31-40.

An additional exemplary probe set includes probes specific for human TOP2A. The probe set includes two or more (such as 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) probes selected from probes including or consisting of the nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% sequence identity with any one of SEQ ID NOs: 41-50 or at least 250 contiguous nucleotides of any one of SEQ ID NOs: 41-50. In one example, a probe set for TOP2A includes probes including or consisting of nucleic acid sequences having at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% sequence identity with each of SEQ ID NOs: 41-50. In a particular example, the probe set includes each of SEQ ID NOs: 41-50.

V. Kits

Also disclosed are kits including one or more of the disclosed nucleic acid probes

(for example, one or more of SEQ ID NOs: 1-50). For example, kits can include at least one probe (such as at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more probes) or at least one probe set (such as at least 1, 2, 3, 4, or 5 probe sets) as described herein. In one example, the kit includes probes including the nucleic acid sequence of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or all of SEQ ID NOs: 1- 10 (or sequences at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% identical to SEQ ID NOs: 1-10) or one or more nucleic acids including at least 250 contiguous nucleotides of any one of sequences at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% identical to SEQ ID NOs: 1-10. In another example, the kit includes probes including the nucleic acid sequence of at least 1, 2, 3, 4, 5, 6, 1, 8, 9, or all of SEQ ID NOs: 1 1 -20 (or sequences at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% identical to SEQ ID NOs: 1 1-20) or one or more nucleic acids including at least 250 contiguous nucleotides of any one of sequences at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% identical to SEQ ID NOs: 1 1-20. In a further example, the kit includes probes including the nucleic acid sequence of at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, or all of SEQ ID NOs: 21-30 (or sequences at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% identical to SEQ ID NOs: 21-30) or one or more nucleic acids including at least 250 contiguous nucleotides of any one of sequences at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% identical to SEQ ID NOs: 21- 30. In a still further example, the kit includes probes including the nucleic acid sequence of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or all of SEQ ID NOs: 31-40 (or sequences at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% identical to SEQ ID NOs: 31-40) or one or more nucleic acids including at least 250 contiguous nucleotides of any one of sequences at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% identical to SEQ ID NOs: 31-40. In an additional example, the kit includes probes including the nucleic acid sequence of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or all of SEQ ID NOs: 41-50 (or sequences at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% identical to SEQ ID NOs: 41-50) or one or more nucleic acids including at least 250 contiguous nucleotides of any one of sequences at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% identical to SEQ ID NOs: 41-50. In some examples, the probes are present in separate containers. In other examples, the probes (or the probe set) are in a single container.

In one particular embodiment, the kit includes ten plasmids, each plasmid including one of SEQ ID NOs: 1-10. The plasmids are pooled and labeled (for example, labeled with DNP) and provided in a vial at a concentration of about 60 μg/ml.

In another particular embodiment, the kit includes ten plasmids, each plasmid including one of SEQ ID NOs: 1 1-20. The plasmids are pooled and labeled (for example, labeled with DNP) and provided in a vial at a concentration of about 60 μg/ml.

In a further particular embodiment, the kit includes ten plasmids, each plasmid including one of SEQ ID NOs: 21-30. The plasmids are pooled and labeled (for example, labeled with DNP) and provided in a vial at a concentration of about 60 μg/ml.

In another particular embodiment, the kit includes ten plasmids, each plasmid including one of SEQ ID NOs: 31-40. The plasmids are pooled and labeled (for example, labeled with DNP) and provided in a vial at a concentration of about 70 μg/ml. In an additional particular embodiment, the kit includes ten plasmids, each plasmid including one of SEQ ID NOs: 41-50. The plasmids are pooled and labeled (for example, labeled with DNP) and provided in a vial at a concentration of about 60 μg/ml.

The kits can also include one or more reagents for detecting a target nucleic acid molecule in a sample (for example, by in situ hybridization or CGH assay), or for producing a detectably labeled probe. For example, a kit can include at least one of the disclosed nucleic acid probes or probe sets, along with one or more buffers, labeled dNTPs, a labeling enzyme (such as a polymerase), primers, nuclease free water, and instructions for producing a labeled probe. In another example, the kit includes one or more of the disclosed nucleic acid probes (unlabeled or labeled) along with buffers and other reagents for performing in situ hybridization. For example, if one or more unlabeled probes are included in the kit, labeling reagents can also be included, along with specific detection agents (for example, fluorescent, chromogenic, luminescent and/or radiometric) and other reagents for performing an in situ hybridization assay, such as paraffin pretreatment buffer, protease(s) and protease buffer, prehybridization buffer, hybridization buffer, wash buffer, counterstain(s), mounting medium, or combinations thereof. In some examples, such kit components are present in separate containers. The kit can optionally further include control slides (such as positive or negative controls, such as those known to contain or not contain the target sequence(s), such as PTEN, PIK3CA, MDM2, MET, or TOP2A nucleic acid sequences) for assessing hybridization and signal of the probe(s).

In certain examples, the kits include avidin, antibodies, and/or receptors (or other anti-ligands). Optionally, one or more of the detection agents (including a primary detection agent, and optionally, secondary, tertiary or additional detection reagents) are labeled, for example, with a hapten or fluorophore (such as a fluorescent dye or quantum dot). In some instances, the detection reagents are labeled with different detectable moieties (for example, different fluorescent dyes, spectrally distinguishable quantum dots, different haptens, etc). For example, a kit can include two or more nucleic acid probes or probe sets that correspond to and are capable of hybridizing to different target nucleic acids (for example, any of the target nucleic acids disclosed herein). The first probe or probe set can be labeled with a first detectable label (e.g., hapten, fluorophore, etc.), the second probe or probe set can be labeled with a second detectable label, and any additional probes or probe sets (e.g., third, fourth, fifth, etc.) can be labeled with additional detectable labels. The first, second, and any subsequent probes or probe sets can be labeled with different detectable labels, although other detection schemes are possible. If the probe(s) are labeled with indirectly detectable labels, such as haptens, the kits can include detection agents (such as labeled avidin, antibodies or other specific binding agents) for some or all of the probes. In one embodiment, the kit includes probes and detection reagents suitable for multiplex ISH.

In one example, the kit also includes an antibody conjugate, such as an antibody conjugated to a label (e.g., an enzyme, fluorophore, or fluorescent nanoparticle). In some examples, the antibody is conjugated to the label through a linker, such as PEG, 6X-His, streptavidin, or GST.

In another example, the kit includes one or more of the disclosed nucleic acid probes affixed to a solid support (such as an array) along with buffers and other reagents for performing CGH. Reagents for labeling sample and control DNA can also be included, along with other reagents for performing an aCGH assay, prehybridization buffer, hybridization buffer, wash buffer, or combinations thereof. The kit can optionally further include control slides for assessing hybridization and signal of the labeled DNAs.

VI. Detectable Labels and Methods of Labeling

The nucleic acid probes disclosed herein can include one or more labels, for example to permit detection of a target nucleic acid molecule. In various applications, such as in situ hybridization procedures, a nucleic acid probe includes a label (e.g., a detectable label). A "detectable label" is a molecule or material that can be used to produce a detectable signal that indicates the presence or concentration of the probe (particularly the bound or hybridized probe) in a sample. Thus, a labeled nucleic acid molecule provides an indicator of the presence or quantity (for example, gene copy number) of a target nucleic acid (to which the labeled uniquely specific nucleic acid molecule is bound or hybridized) in a sample. The disclosure is not limited to the use of particular labels, although examples are provided.

A label associated with one or more nucleic acid molecules (such as the disclosed probes) can be detected either directly or indirectly. A label can be detected by any known or yet to be discovered mechanism including absorption, emission and/or scattering of a photon (including radio frequency, microwave frequency, infrared frequency, visible frequency and ultra-violet frequency photons). Detectable labels include colored, fluorescent, phosphorescent and luminescent molecules and materials, catalysts (such as enzymes) that convert one substance into another substance to provide a detectable difference (such as by converting a colorless substance into a colored substance or vice versa, or by producing a precipitate or increasing sample turbidity), haptens that can be detected by antibody binding interactions, and paramagnetic and magnetic molecules or materials.

Particular examples of detectable labels include fluorescent molecules (or fluorochromes). Numerous fluorochromes are known to those of skill in the art, and can be selected, for example from Life Technologies, e.g., see, The Handbook— A Guide to

Fluorescent Probes and Labeling Technologies. Examples of particular fluorophores that can be attached (for example, chemically conjugated) to a nucleic acid molecule (such as a uniquely specific binding region) are provided in U.S. Patent No. 5,866,366 to Nazarenko et al, such as 4-acetamido-4'-isothiocyanatostilbene-2,2'disulfonic acid, acridine and derivatives such as acridine and acridine isothiocyanate, 5-(2'- aminoethyl)aminonaphthalene- l -sulfonic acid (EDANS), 4-amino-N-[3- vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate (Lucifer Yellow VS), N-(4-anilino- 1 - naphthyl)maleimide, anthranilamide, Brilliant Yellow, coumarin and derivatives such as coumarin, 7-amino-4-methylcoumarin (AMC, Coumarin 120), 7-amino-4- trifluoromethylcouluarin (Coumarin 151); cyanosine; 4',6-diaminidino-2-phenylindole

(DAPI); 5', 5"-dibromopyrogallol-sulfonephthalein (Bromopyrogallol Red); 7- diethylamino-3-(4'-isothiocyanatophenyl)-4-methylcoumarin; diethylenetriamine pentaacetate; 4,4'-diisothiocyanatodihydro-stilbene-2,2'-disulfonic acid; 4,4'- diisothiocyanatostilbene-2,2'-disulfonic acid; 5-[dimethylamino]naphthalene- l -sulfonyl chloride (DNS, dansyl chloride); 4-(4'-dimethylaminophenylazo)benzoic acid (DABCYL);

4-dimethylaminophenylazophenyl-4' -isothiocyanate (DABITC); eosin and derivatives such as eosin and eosin isothiocyanate; erythrosin and derivatives such as erythrosin B and erythrosin isothiocyanate; ethidium; fluorescein and derivatives such as 5- carboxyfluorescein (FAM), 5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF), 2'7'- dimethoxy-4'5'-dichloro-6-carboxyfluorescein (JOE), fluorescein, fluorescein

isothiocyanate (FITC), and QFITC (XRITC); 2', 7'-difluorofluorescein (OREGON

GREEN®); fluorescamine; IR144; IR1446; Malachite Green isothiocyanate; 4- methylumbelliferone; ortho-cresolphthalein; nitrotyrosine; pararosaniline; Phenol Red; B- phycoerythrin; o-phthaldialdehyde; pyrene and derivatives such as pyrene, pyrene butyrate and succinimidyl 1 -pyrene butyrate; Reactive Red 4 (Cibacron Brilliant Red 3B-A);

rhodamine and derivatives such as 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissamine rhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine X isothiocyanate, rhodamine green, sulforhodamme B, sulforhodamme 101 and sulfonyl chloride derivative of sulforhodamme 101 (Texas Red); N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA); tetramethyl rhodamine; tetramethyl rhodamine isothiocyanate (TRITC); riboflavin; rosolic acid and terbium chelate derivatives.

Other suitable fluorophores include thiol-reactive europium chelates which emit at approximately 617 nm (Heyduk and Heyduk, Analyt. Biochem. 248:216-27, 1997; J. Biol. Chem. 274:3315-22, 1999), as well as GFP, Lissamine™, diethylaminocoumarin, fluorescein chlorotriazinyl, naphthofluorescein, 4,7-dichlororhodamine and xanthene (as described in U.S. Patent No. 5,800,996 to Lee et al.) and derivatives thereof. Other fluorophores known to those skilled in the art can also be used, for example those available from Life Technologies (Carlsbad, CA) and including the ALEXA FLUOR® series of dyes (for example, as described in U.S. Patent Nos. 5,696,157, 6, 130, 101 and 6, 716,979), the BODIPY series of dyes (dipyrrometheneboron difluoride dyes, for example as described in U.S. Patent Nos. 4,774,339, 5, 187,288, 5,248,782, 5,274,1 13, 5,338,854, 5,451,663 and 5,433,896), Cascade Blue (an amine reactive derivative of the sulfonated pyrene described in U.S. Patent No. 5, 132,432) and Marina Blue (U.S. Patent No.

5,830,912).

In addition to the fluorochromes described above, a fluorescent label can be a fluorescent nanoparticle, such as a semiconductor nanocrystal, e.g., a quantum dot (obtained, for example, from Life Technologies); see also, U.S. Patent Nos. 6,815,064; 6,682596; and 6,649,138). Semiconductor nanocrystals are microscopic particles having size-dependent optical and/or electrical properties. When semiconductor nanocrystals are illuminated with a primary energy source, a secondary emission of energy occurs of a frequency that corresponds to the bandgap of the semiconductor material used in the semiconductor nanocrystal. This emission can be detected as colored light of a specific wavelength or fluorescence. Semiconductor nanocrystals with different spectral characteristics are described in e.g., U.S. patent No. 6,602,671. Semiconductor nanocrystals that can be coupled to a variety of biological molecules (including dNTPs and/or nucleic acids) or substrates by techniques described in, for example, Bruchez et al, Science 281 :2013-2016, 1998; Chan et a/., Science 281 :2016-2018, 1998; and U.S. Patent No. 6,274,323.

Formation of semiconductor nanocrystals of various compositions are disclosed in, e.g., U.S. Patent Nos. 6,927,069; 6,914,256; 6,855,202; 6,709,929; 6,689,338; 6,500,622; 6,306,736; 6,225,198; 6,207,392; 6,1 14,038; 6,048,616; 5,990,479; 5,690,807; 5,571,018; 5,505,928; 5,262,357 and in U.S. Patent Publication No. 2003/0165951 as well as PCT Publication No. WO 99/26299. Separate populations of semiconductor nanocrystals can be produced that are identifiable based on their different spectral characteristics. For example, semiconductor nanocrystals can be produced that emit light of different colors based on their composition, size or size and composition. For example, quantum dots that emit light at different wavelengths based on size (565 nm, 655 nm, 705 nm, or 800 nm emission wavelengths), which are suitable as fluorescent labels in the probes disclosed herein are available from Life Technologies (Carlsbad, CA).

Additional labels include, for example, radioisotopes (such as³H), metal chelates such as DOTA and DPTA chelates of radioactive or paramagnetic metal ions like Gd³⁺, and liposomes.

Detectable labels that can be used with nucleic acid molecules (such as the disclosed probes) also include enzymes, for example horseradish peroxidase (HRP), alkaline phosphatase (AP), acid phosphatase, glucose oxidase, β-galactosidase, β- glucuronidase, or β-lactamase. Where the detectable label includes an enzyme, a chromogen, fluorogenic compound, or luminogenic compound can be used in combination with the enzyme to generate a detectable signal (numerous of such compounds are commercially available, for example, from Life Technologies). Particular examples of chromogenic compounds include diaminobenzidine (DAB), 4-nitrophenylphosphate (pNPP), fast red, fast blue, bromochloroindolyl phosphate (BCIP), nitro blue tetrazolium (NBT), BCIP/NBT, AP Orange, AP blue, tetramethylbenzidine (TMB), 2,2'-azino-di-[3- ethylbenzothiazoline sulphonate] (ABTS), o-dianisidine, 4-chloronaphthol (4-CN), nitrophenyl- -D-galactopyranoside (ONPG), o-phenylenediamine (OPD), 5-bromo-4- chloro-3-indolyl- -galactopyranoside (X-Gal), methylumbelliferyl- -D-galactopyranoside (MU-Gal), p-nitrophenyl-a-D-galactopyranoside (PNP), 5-bromo-4-chloro-3-indolyl- β - D-glucuronide (X-Gluc), 3-amino-9-ethyl carbazol (AEC), fuchsin, iodonitrotetrazolium (INT), tetrazolium blue, and tetrazolium violet.

Alternatively, an enzyme can be used in a metallographic detection scheme. For example, silver in situ hybridization (SISH) procedures involve metallographic detection schemes for identification and localization of a hybridized genomic target nucleic acid sequence. Metallographic detection methods include using an enzyme, such as alkaline phosphatase, in combination with a water-soluble metal ion and a redox-inactive substrate of the enzyme. The substrate is converted to a redox-active agent by the enzyme, and the redox-active agent reduces the metal ion, causing it to form a detectable precipitate. (See, for example, U.S. Patent Application Publication No. 2005/0100976, PCT Publication No. 2005/003777 and U.S. Patent Application Publication No. 2004/0265922). Metallographic detection methods also include using an oxido-reductase enzyme (such as horseradish peroxidase) along with a water soluble metal ion, an oxidizing agent and a reducing agent, again to form a detectable precipitate. (See, for example, U.S. Patent No. 6,670, 113).

In non-limiting examples, the disclosed nucleic acid probes are labeled with dNTPs covalently attached to hapten molecules (such as a nitro-aromatic compound (e.g., 2,4-dinitrophenyl (DNP)), biotin, fluorescein, digoxigenin, etc.). Additional haptens suitable for labeling the disclosed probes include nitropyrazole, 3-hydroxyquinoxaline, thiazolesulfonamide, nitrocinnamic acid, rotenone, 7-(diethylamino)coumarin-3-carboxylic acid, benzodiazepine, or benzofuran haptens (see, e.g., International Pat. Publ. No. WO 2012/003476. incorporated herein by reference). Methods for conjugating haptens and other labels to dNTPs (e.g., to facilitate incorporation into labeled probes) are well known in the art. For examples of procedures, see, e.g., U.S. Patent Nos. 5,258,507, 4,772,691, 5,328,824, and 4,71 1,955. Indeed, numerous labeled dNTPs are available commercially, for example from Life Technologies (Carlsbad, CA). A label can be directly or indirectly attached to a dNTP at any location on the dNTP, such as a phosphate (e.g., a, β or γ phosphate) or a sugar.

Detection of labeled nucleic acid molecules can be accomplished by contacting the hapten-labeled nucleic acid molecules bound to the genomic target nucleic acid with a primary anti-hapten antibody. In one example, the primary anti-hapten antibody (such as a mouse anti-hapten antibody) is directly labeled with an enzyme. In another example, a secondary anti-antibody (such as a goat anti-mouse IgG antibody) conjugated to an enzyme is used for signal amplification. In CISH a chromogenic substrate is added, for SISH, silver ions and other reagents as outlined in the referenced patents/applications are added.

In some examples, a probe is labeled by incorporating one or more labeled dNTPs using an enzymatic (polymerization) reaction. For example, the disclosed nucleic acid probes (for example, incorporated into a plasmid vector) can be labeled by nick translation (using, for example, biotin, DNP, digoxigenin, etc.) or by random primer extension with terminal transferase (e.g., 3' end tailing). In some examples, the nucleic probe is labeled by a modified nick translation reaction where the ratio of DNA polymerase I to deoxyribonuclease I (DNase I) is modified to produce greater than 100% of the starting material. In particular examples, the nick translation reaction includes DNA polymerase I to DNase I at a ratio of at least about 800: 1, such as at least 2000: 1, at least 4000: 1, at least 8000: 1, at least 10,000: 1, at least 12,000: 1, at least 16,000: 1, such as about 800: 1 to 24,000: 1 and the reaction is carried out overnight (for example, for about 16-22 hours) at a substantially isothermal temperature, for example, at about 16°C to 25°C (such as room temperature). If the probe is included in a probe set (for example, multiple plasmids, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, or more plasmids), the plasmids may be mixed in an equal molar ratio prior to performing the labeling reaction (such as nick translation or modified nick translation).

In other examples, chemical labeling procedures can also be employed. Numerous reagents (including hapten, fluorophore, and other labeled nucleotides) and other kits are commercially available for enzymatic labeling of nucleic acids, including the disclosed nucleic acid probes. As will be apparent to those of skill in the art, any of the labels and detection procedures disclosed above are applicable in the context of labeling a probe, e.g., for use in in situ hybridization reactions. For example, the Amersham MULTIPRIME® DNA labeling system, various specific reagents and kits available from Molecular Probes/Life Technologies, or any other similar reagents or kits can be used to label the nucleic acids disclosed herein. In particular examples, the disclosed probes can be directly or indirectly labeled with a hapten, a ligand, a fluorescent moiety (e.g., a fluorophore or a semiconductor nanocrystal), a chromogenic moiety, or a radioisotope. For example, for indirect labeling, the label can be attached to nucleic acid molecules via a linker (e.g., PEG or biotin). Additional methods that can be used to label probe nucleic acid molecules are provided in U.S. Application Pub. No. 2005/0158770.

VI. Methods of Detecting a Target Nucleic Acid

The disclosed probes can be used for nucleic acid detection, such as ISH procedures (for example, fluorescence in situ hybridization (FISH), chromogenic in situ hybridization (CISH) and silver in situ hybridization (SISH)) or comparative genomic hybridization (CGH). Exemplary uses are discussed below.

In some embodiments, the disclosed methods of detecting a target nucleic acid include comparing the signal or gene copy number detected in a sample utilizing one or more of the disclosed probes or probe sets in a sample with a control or reference value. In some examples, a change in signal from a probe or probe set relative to a control (such as an increase of about 1.5-fold, 2-fold, 3-fold, 5-fold, 10-fold, 20-fold, or more relative to a control sample or reference value) indicates the presence, expression, or gene copy number of a target nucleic acid (such as PTEN, PIK3CA, MDM2, MET, and/or TOP2A) in the sample. In some examples, change in expression of a target nucleic acid (such as PTEN, PIK3CA, MDM2, MET, and/or TOP2A) compared to a control (such as an increase or decrease of about 1.5-fold, 2-fold, 3-fold, 5-fold, 10-fold, 20-fold, or more relative to a control sample or reference value) indicates presence or prognosis of a tumor. In other examples, the gene copy number of a target nucleic acid (such as PTEN, PIK3CA, MDM2, MET, and/or TOP2A) is determined and an increase in gene copy number (such as a gene copy number greater than about 2, 3, 4, 5, 10, 20, or more) indicates the presence or prognosis of a tumor.

In other examples, the probes may be used to analyze copy number changes associated with autism (e.g., cultured peripheral blood lymphocytes as described by van Daalen et al, Neurogenetics 12:315-323, 2011). In another example, the probes may be used to evaluate signaling pathways associated with leptins and metabolism (e.g., Donato et al., Arq. Bras. Endocrinol. Metab. 54(7):591-602, 2010 and Williams et al, J. Neurosci. 31(37): 13147- 13156, 201 1).

A. In Situ Hybridization

In situ hybridization (ISH) involves contacting a sample containing a target nucleic acid (e.g., a genomic target nucleic acid) in the context of a metaphase or interphase chromosome preparation (such as a cell or tissue sample mounted on a slide) with a labeled probe specifically hybridizable or specific for the target nucleic acid (for example, one or more of the probes disclosed herein). The slides are optionally pretreated, e.g., to remove paraffin or other materials that can interfere with uniform hybridization. The chromosome sample and the probe are both treated, for example by heating to denature the double stranded nucleic acids. The probe (formulated in a suitable hybridization buffer) and the sample are combined, under conditions and for sufficient time to permit hybridization to occur (typically to reach equilibrium). The chromosome preparation is washed to remove excess probe, and detection of specific labeling of the target is performed using standard techniques.

For example, a biotinylated probe can be detected using fluorescein-labeled avidin or avidin-alkaline phosphatase. For fluorochrome detection, the fluorochrome can be detected directly, or the samples can be incubated, for example, with fluorescein isothiocyanate (FITC)-conjugated avidin. Amplification of the FITC signal can be effected, if necessary, by incubation with biotin-conjugated goat anti-avidin antibodies, washing and a second incubation with FITC-conjugated avidin. For detection by enzyme activity, samples can be incubated, for example, with streptavidin, washed, incubated with biotin-conjugated alkaline phosphatase, washed again and pre- equilibrated (e.g., in alkaline phosphatase (AP) buffer). The enzyme reaction can be performed in, for example, AP buffer containing NBT/BCIP and stopped by incubation in 2 X SSC. For a general description of in situ hybridization procedures, see, e.g., U.S. Patent No. 4,888,278. Numerous procedures for FISH, CISH, and SISH are known in the art. For example, procedures for performing FISH are described in U.S. Patent Nos. 5,447,841 ; 5,472,842; and 5,427,932; and for example, in Pinkel et al, Proc. Natl. Acad. Sci.

83:2934-2938, 1986; Pinkel et al, Proc. Natl. Acad. Sci. 85:9138-9142, 1988; and Lichter et al, Proc. Natl. Acad. Sci. 85:9664-9668, 1988. CISH is described in, e.g., Tanner et al,

Am. J. Pathol. 157: 1467- 1472, 2000 and U.S. Patent No. 6,942,970. Additional detection methods are provided in U.S. Patent No. 6,280,929.

Numerous reagents and detection schemes can be employed in conjunction with FISH, CISH, and SISH procedures to improve sensitivity, resolution, or other desirable properties. As discussed above, probes labeled with fluorophores (including fluorescent dyes and quantum dots) can be directly optically detected when performing FISH.

Alternatively, the probe can be labeled with a non- fluorescent molecule, such as a hapten (such as the following non-limiting examples: biotin, digoxigenin, DNP, and various oxazoles, pyrrazoles, thiazoles, nitroaryls, benzofurazans, triterpenes, ureas, thioureas, rotenones, coumarin, courmarin-based compounds, Podophyllotoxin, Podophyllotoxin- based compounds, and combinations thereof), ligand or other indirectly detectable moiety. Probes labeled with such non- fluorescent molecules (and the target nucleic acid sequences to which they bind) can then be detected by contacting the sample (e.g., the cell or tissue sample to which the probe is bound) with a labeled detection reagent, such as an antibody (or receptor, or other specific binding partner) specific for the chosen hapten or ligand.

The detection reagent can be labeled with a fluorophore (e.g., quantum dot) or with another indirectly detectable moiety, or can be contacted with one or more additional specific binding agents (e.g., secondary or specific antibodies), which can in turn be labeled with a fluorophore. Optionally, the detectable label is attached directly to the antibody, receptor (or other specific binding agent). Alternatively, the detectable label is attached to the binding agent via a linker, such as a hydrazide thiol linker, a polyethylene glycol linker, or any other flexible attachment moiety with comparable reactivities. For example, a specific binding agent, such as an antibody, a receptor (or other anti-ligand), avidin, or the like can be covalently modified with a fluorophore (or other label) via a heterobifunctional polyalkyleneglycol linker such as a heterobifunctional polyethyleneglycol (PEG) linker. A heterobifunctional linker combines two different reactive groups selected, e.g., from a carbonyl-reactive group, an amine-reactive group, a thiol-reactive group and a photo- reactive group, the first of which attaches to the label and the second of which attaches to the specific binding agent. In other examples, the probe, or specific binding agent (such as an antibody, e.g., a primary antibody, receptor or other binding agent) is labeled with an enzyme that is capable of converting a fluorogenic or chromogenic composition into a detectable fluorescent, colored or otherwise detectable signal (e.g., as in deposition of detectable metal particles in SISH). As indicated above, the enzyme can be attached directly or indirectly via a linker to the relevant probe or detection reagent. Examples of suitable reagents (e.g., binding reagents) and chemistries (e.g., linker and attachment chemistries) are described in U.S. Patent Application Publication Nos. 2006/0246524; 2006/0246523, and 2007/01 17153.

In further examples, a signal amplification method is utilized, for example, to increase sensitivity of the probe. For example, CAtalyzed Reporter Deposition (CARD), also known as Tyramide Signal Amplification (TSA™) may be utilized. In one variation of this method a biotinylated nucleic acid probe detects the presence of a target by binding thereto. Next a streptavidin-peroxidase conjugate is added. The streptavidin binds to the biotin. A substrate of biotinylated tyramide (tyramine is 4-(2- aminoethyl)phenol) is used, which presumably becomes a free radical when interacting with the peroxidase enzyme. The phenolic radical then reacts quickly with the surrounding material, thus depositing or fixing biotin in the vicinity. This process is repeated by providing more substrate (biotinylated tyramide) and building up more localized biotin. Finally, the "amplified" biotin deposit is detected with streptavidin attached to a fluorescent molecule.

Alternatively, the amplified biotin deposit can be detected with avidin-peroxidase complex, that is then fed 3,3'-diaminobenzidine to produce a brown color. It has been found that tyramide attached to fluorescent molecules also serve as substrates for the enzyme, thus simplifying the procedure by eliminating steps.

In other examples, the signal amplification method utilizes branched DNA (bDNA) signal amplification. In some examples, target-specific oligonucleotides (label extenders and capture extenders) are hybridized with high stringency to the target nucleic acid. Capture extenders are designed to hybridize to the target and to capture probes, which are attached to a microwell plate. Label extenders are designed to hybridize to contiguous regions on the target and to provide sequences for hybridization of a preamplifier oligonucleotide. Signal amplification then begins with preamplifier probes hybridizing to label extenders. The preamplifier forms a stable hybrid only if it hybridizes to two adjacent label extenders. Other regions on the preamplifier are designed to hybridize to multiple bDNA amplifier molecules that create a branched structure. Finally, alkaline phosphatase (AP)-labeled oligonucleotides, which are complementary to bDNA amplifier sequences, bind to the bDNA molecule by hybridization. The bDNA signal is the chemiluminescent product of the AP reaction See, e.g., Tsongalis, Microbiol. Inf. Dis. 126:448-453, 2006; U.S. Pat. No. 7,033,758.

In further examples, the signal amplification method utilizes polymerized antibodies. In some examples, the labeled probe is detected by using a primary antibody to the label (such as an anti-DIG or anti-DNP antibody). The primary antibody is detected by a polymerized secondary antibody (such as a polymerized HRP-conjugated secondary antibody or an AP-conjugated secondary antibody). The enzymatic reaction of AP or HRP leads to the formation of strong signals that can be visualized.

It will be appreciated by those of skill in the art that by appropriately selecting labeled probe-specific binding agent pairs, multiplex detection schemes can be produced to facilitate detection of multiple target nucleic acids (e.g., genomic target nucleic acids) in a single assay (e.g., on a single cell or tissue sample or on more than one cell or tissue sample). For example, a first probe that corresponds to a first target nucleic acid can be labeled with a first hapten, such as biotin, while a second probe that corresponds to a second target nucleic acid can be labeled with a second hapten, such as DNP. Following exposure of the sample to the probes, the bound probes can be detected by contacting the sample with a first specific binding agent (in this case avidin labeled with a first fluorophore, for example, a first spectrally distinct quantum dot, e.g., that emits at 585 nm) and a second specific binding agent (in this case an anti-DNP antibody, or antibody fragment, labeled with a second fluorophore (for example, a second spectrally distinct quantum dot, e.g., that emits at 705 nm). Additional probes/binding agent pairs can be added to the multiplex detection scheme using other spectrally distinct fluorophores. Numerous variations of direct, and indirect (one step, two step or more) can be envisioned, all of which are suitable in the context of the disclosed probes and assays.

Additional details regarding certain detection methods, e.g., as utilized in CISH and SISH procedures, can be found in Bourne, The Handbook of Immunoper oxidase Staining Methods , published by Dako Corporation, Santa Barbara, CA.

In some embodiments, the disclosed probes can be used in methods of determining the copy number of a target nucleic acid (such as PTEN, PIK3CA, MDM2, MET, or TOP2A) in a biological sample (such as a tissue sample). Methods of determining the copy number of a gene or chromosomal region are well known to those of skill in the art. In some examples, the methods include in situ hybridization (such as fluorescent, chromogenic, or silver in situ hybridization), comparative genomic hybridization, or polymerase chain reaction (such as real-time quantitative PCR). In some examples, methods of determining gene copy number include counting the number of ISH signals (such as fluorescent, colored, or silver spots) for the target nucleic acid in one or more individual cells. The methods may also include counting the number of ISH signals (such as fluorescent, colored, or silver spots) for a reference (such as a chromosome-specific probe) in the cells. In particular examples, the number of copies of the gene (or chromosome) may be estimated by the person (or computer, in the case of an automated method) scoring the slide. In some examples, an increased copy number relative to a control (such as an increase of about 1.5-fold, 2-fold, 3-fold, 5-fold, 10-fold, 20-fold, or more relative to a control sample or reference value) indicates an increase in the target nucleic acid copy number.

In some examples, the method includes counting the number of copies per cell or nucleus of a reference, such as a chromosomal locus known not to be abnormal, for example a centromere. In some examples, the reference is on the same chromosome as the gene of interest. Exemplary reference chromosomes that can be used for particular human genes of interest are provided in Table 1. In particular examples, the reference locus is detected by using a centromere-specific probe. Such probes are known in the art and are commercially available, for example, Vysis CEP probes (Abbott Molecular, Des Plaines, IL) and SPOTLIGHT centromeric probes (Invitrogen, Carlsbad, CA). In some examples, a ratio of target nucleic acid copy number to reference copy number greater than about two (such as greater than about 2, 3, 4, 5, 10, 20, or more), indicates an increase in the target nucleic acid copy number.

Table 1. Exemplary reference chromosomes for particular target nucleic acids

B. Microarray Applications

Comparative genomic hybridization (CGH) is a molecular-cytogenetic method for the analysis of copy number changes (gain/loss) in the DNA content of cells. The contribution of genome structural variation to human disease is found in rare genomic disorders (for example, Trisomy 21 , Prader-Willi Syndrome) and a broad range of human diseases, such as genetic diseases, autism, schizophrenia, cancers, and autoimmune diseases. In one example, the method is based on the hybridization of differently fluorescently labeled sample DNA (for example, labeled with fluorescein-FITC) and normal DNA (for example, labeled with rhodamine or Texas red) to normal human metaphase preparations. Using methods known in the art, such as epifluorescence microscopy and quantitative image analysis, regional differences in the fluorescence ratio of sample versus control DNA can be detected and used for identifying abnormal regions in the sample cell genome. CGH detects unbalanced chromosomes changes (such as increase or decrease in DNA copy number). See, e.g., Kallioniemi et al, Science 258:818- 821, 1992; U.S. Pat. Nos. 5,665,549 and 5,721,098.

Genomic DNA copy number may also be determined by array CGH (aCGH). See, e.g., Pinkel and Albertson, Nat. Genet. 37:S 11-S17, 2005; Pinkel et al, Nat. Genet.

20:207-211, 1998; Pollack et al, Nat. Genet. 23:41-46, 1999. Similar to standard CGH, sample and reference DNA are differentially labeled and mixed. However, for aCGH, the DNA mixture is hybridized to a slide containing hundreds or thousands of defined DNA probes (such as probes that specifically hybridize to a genomic target nucleic acid of interest). The fluorescence intensity ratio at each probe in the array is used to evaluate regions of DNA gain or loss in the sample, which can be mapped in finer detail than CGH, based on the particular probes which exhibit altered fluorescence intensity.

In general, CGH (and aCGH) does not provide information as to the exact number of copies of a particular genomic DNA or chromosomal region. Instead, CGH provides information on the relative copy number of one sample (such as a tumor sample) compared to another (such as a reference sample, for example a non-tumor cell or tissue sample). Thus, CGH is most useful to determine whether genomic DNA copy number of a target nucleic acid is increased or decreased as compared to a reference sample thereby determining the copy number variation of a target nucleic acid sample relative to a reference sample.

In a particular example, the disclosed probes may be utilized for aCGH. For example, an unlabeled probe disclosed herein (such as one or more of any one of SEQ ID NOs: 1-50 or a portion thereof) may be immobilized on a solid surface (such as nitrocellulose, nylon, glass, cellulose acetate, plastics (for example, polyethylene, polypropylene, or polystyrene), paper, ceramics, metals, and the like). Methods of immobilizing nucleic acids on a solid surface are well known in the art (see, e.g., Bischoff et αΙ, ΑηαΙ Biochem. 164:336-344, 1987; Kremsky et al, Nuc. Acids Res. 15:2891-2910, 1987). As discussed above, differently fluorescently labeled sample DNA (for example, labeled with fluorescein-FITC) and reference DNA (for example, labeled with rhodamine or Texas red) is hybridized to the probe array and regional differences in the fluorescence ratio of sample versus reference DNA can be detected and used for identifying abnormal regions in the sample cell genome.

In another example, disclosed probes are synthesized in situ on a solid surface (such as nitrocellulose, nylon, glass, cellulose acetate, plastics (for example, polyethylene, polypropylene, or polystyrene), paper, ceramics, metals, and the like). For example, the probes are utilized for printing, in situ, on a solid support utilizing computer based microarray printing methodologies, such as those described in U.S. Pat. Nos. 6,315,958; 6,444, 175; and 7,083,975 and U.S. Pat. Application Nos. 2002/0041420, 2004/0126757, 2007/0037274, and 2007/0140906. In some examples, using a maskless array synthesis (MAS) instrument, oligonucleotides synthesized in situ on the microarray are under software control resulting in individually customized arrays based on the particular needs of an investigator. The number of probes synthesized on a microarray varies, for example presently anywhere from 50,000 to 2.1 million probes, in various configurations, can be synthesized on a single microarray slide (for example, Roche NimbleGen CGH

microarrays contain from 385,000 to 4 million or more probes/array).

Probe sequences are synthesized either in situ by MAS instruments, or alternatively by utilizing photolithographic methods as described in U.S. Pat. Nos.

5,143,854; 5,424, 186; 5,405,783; and 5,445,934. Utilizing the disclosed probes for microarray applications is not limited by their method of manufacture, and a skilled artisan will understand additional methods of creating microarrays with uniquely specific oligonucleotide probes thereon that are equally applicable. For example, historical methods of spotting nucleic acid sequences onto solid supports are also contemplated, such that historically utilized nucleic acid probes are replaced by uniquely specific probes as described herein. Regardless of method used to place probes on a microarray, the uniquely specific probes can be used to target one or more nucleic acid samples, either individually or on the same array.

Applications of the probes disclosed herein that are in situ synthesized or otherwise immobilized on a microarray slide can be utilized for aCGH as well as other microarray based genomic target enrichment applications such as those described in U.S. Pat. Publication Nos. 2008/0194413, 2008/0194414, 2009/0203540, and 2009/0221438. Utilizing uniquely specific probes for generating in situ synthesized microarrays provides many improvements over current microarray probe designs. For example, use of uniquely specific probes allows for more specific binding of target sequences as compared to current probes, therefore not as many probes are needed per target and/or in conjunction more can be added to capture additional targets. Further, the need for blocking DNA (for example, Cot-1™ DNA) typically utilized in micro-array experiments is reduced or eliminated when utilizing uniquely specific oligonucleotide probes.

For CGH applications, typically both target and reference genomic DNA are hybridized on one array for comparison on one microarray substrate. The CGH Analysis User's Guide (version 5.1, Roche NimbleGen, Madison, WI; available on the World Wide Web at nimblegen.com) describes methods for performing CGH analysis utilizing microarrays. In general, two genomic DNA samples, a target sample and a reference sample, are fragmented and labeled with different detection moieties (for example, Cy-3 and Cy-5 fluorescent moieties). The two labeled samples are mixed and hybridized to a microarray support, in this case a microarray comprising uniquely specific oligonucleotide probes, and the microarray is subsequently assayed for both detection moieties. The microarrays are scanned and detection data captured, for example by scanning a microarray with a microarray scanner (for example, a MS200 Microarray Scanner; Roche NimbleGen). The data is analyzed using analysis software (for example, NimbleScan; Roche NimbleGen). The target genomic sequence data is compared to the reference and DNA copy number gains and losses in target samples are thereby characterized. The target genomic sequences can be, for example, from targeted region(s) of one or more chromosome(s), one whole chromosome, or the total genomic complement of an organism (for example, a eukaryotic genome, such as a mammalian genome, for example a human genome).

For genomic enrichment (also known as sequence capture), typically a genomic sample is hybridized to a microarray support comprising targeted sequence specific probes for specific target enrichment prior to downstream applications, such as sequencing. The Sequence Capture User's Guide (version 3.1, Roche NimbleGen, incorporated by reference herein) describes methods for performing genomic enrichment. In general, a genomic DNA sample is prepared for hybridization to a microarray support, in this case a microarray comprising the disclosed uniquely specific oligonucleotide probes designed to capture targeted sequences from a genomic sample for enrichment. The captured genomic sequences are then eluted from the microarray support and sequenced, or used for other applications.

C. Blocking DNA

Genome-specific blocking DNA (such as human DNA, for example, total human placental DNA or Cot-1™ DNA) is usually included in a hybridization solution (such as for in situ hybridization) to suppress probe hybridization to repetitive DNA sequences or to counteract probe hybridization to highly homologous (frequently identical) off target sequences when a probe complementary to a human genomic target nucleic acid is utilized. In hybridization with standard probes, in the absence of genome-specific blocking DNA, an unacceptably high level of background staining (for example, non-specific binding, such as hybridization to non-target nucleic acid sequence) is usually present, even when a "repeat- free" probe is used. The disclosed nucleic acid probes exhibit reduced background staining, even in the absence of blocking DNA. In particular examples, the hybridization solution including the disclosed probes does not include genome-specific blocking DNA (for example, total human placental DNA or Cot- 1™ DNA, if the probe is complementary to a human genomic target nucleic acid). This advantage is derived from the uniquely specific nature of the target sequences included in the nucleic acid probe; each labeled probe sequence binds only to the cognate uniquely specific genomic sequence. This results in dramatic increases in signal to noise ratios for ISH techniques.

In some examples the hybridization solution may contain carrier DNA from a different organism (for example, salmon sperm DNA or herring sperm DNA, if the genomic target nucleic acid is a human genomic target nucleic acid) to reduce non-specific binding of the probe to non-DNA materials (for example to reaction vessels or slides) with high net positive charge which can non-specifically bind to the negatively charged probe DNA.

The disclosure is further illustrated by the following non-limiting Examples.

EXAMPLES

Example 1

ISH Detection of PTEN, PIK3CA, MDM2, MET, and TOP2A

This example describes detection of target nucleic acids in tissue samples by ISH using the disclosed PTEN, PIK3CA, MDM2, MET, and TOP2A probes.

Probes were labeled with DNP for ISH. For each target nucleic acid (PIK3CA, PTEN, TOP2A, MET, or MDM2), a set of 10 plasmids including the appropriate probe sequences (PIK3CA - SEQ ID NOs: 1 1-20; PTEN - SEQ ID NOs: 1-10; TOP2A - SEQ ID NOs: 41-50; MET - SEQ ID NOs: 31-40; MDM2 - SEQ ID NOs: 21-30) were pooled, nick translation labeled with DNP, and bulked in Hybrisol® and TE. FFPE tissues were processed for ISH using a BENCHMARK ULTRA system (Ventana Medical Systems, Tucson, AZ) with the conditions for each probe as shown in Table 2. In addition to the target nucleic acid probe, the ISH reactions also each included a centromere probe for the chromosome where the target nucleic acid is located (e.g., PIK3CA - chromosome 3; PTEN - chromosome 10; TOP2A - chromosome 17; MET - chromosome 7; MDM2 - chromosome 12).

All gene probes were labeled with DNP and all reference (centromere) probes were labeled with DIG. DNP-labeled probes were detected using SISH, which produces black staining. DIG-labeled probes were detected using a red chromogen. These were carried out with the following reagents: ultraView™ Red ISH DIG Detection Kit (Ventana Medical Systems) and ultraView™ SISH DNP Detection Kit (Ventana Medical Systems).

Table 2. ISH conditions

Representative digital images of the ISH reactions are shown in FIGS. 1-5. Each probe provided sensitive and specific staining results (Table 3). Tissue samples exhibiting the expected readable results (by a qualified reader; e.g., an appropriately trained pathologist) were designated as "pass." Factors that may contribute to readability include: sufficiently dark staining so as to be apparent, staining across the entire tissue such that all regions of the tissue exhibit the expected result, low background so as to eliminate detecting a false signal, and the biological meaning conferred by the signal matching the expected signal. The characteristics of the signal may vary according to tissue type. Table 3. ISH results

Example 2

Detection of a Target Nucleic Acid

This example describes particular methods that can be used for detecting a target nucleic acid in a sample utilizing probes described herein. However, one skilled in the art will appreciate that methods that deviate from these specific methods can also be used to successfully detect a target nucleic acid.

A sample, such as a tissue sample, is obtained from a subject. Tissue samples are prepared for ISH, including deparaffinization and protease digestion. The sample, such as a tissue or cell sample present on a substrate (such as a microscope slide) is incubated with a PTEN probe set (for example, a probe set including each of SEQ ID NOs: 1-10). The hybridization is carried out in the absence of human DNA blocking reagent (for example, in the absence of Cot-1™ DNA). Hybridization of the PTEN probe set to the sample is detected, for example, using microscopy. Detection of hybridization indicates presence of PTEN nucleic acid in the sample. In some examples, gene copy number is detected.

One of skill in the art will understand that similar methods can be used to detect the presence of other target nucleic acids in a sample. For example, a probe set (for example, a probe set including each of SEQ ID NOs: 1 1-20) may be used to detect presence of PIK3CA in a sample, a probe set (for example, a probe set including each of SEQ ID NOs: 21-30) may be used to detect presence of MDM2 in a sample, a probe set (for example, a probe set including each of SEQ ID NOs: 31-40) may be used to detect presence of MET in a sample, or a probe set (for example, a probe set including each of SEQ ID NOs: 41-50) may be used to detect presence of TOP2A in a sample,

In view of the many possible embodiments to which the principles of the disclosure may be applied, it should be recognized that the illustrated embodiments are only examples and should not be taken as limiting the scope of the invention. Rather, the scope of the invention is defined by the following claims. We therefore claim invention all that comes within the scope and spirit of these claims.

Claims

PATENT CLAIMS

1. An isolated nucleic acid probe comprising:

(a) a nucleic acid molecule having at least 90% sequence identity with the sequence according to any one of SEQ ID NOs: 1-50; or

(b) a nucleic acid molecule having at least 90% sequence identity with at least 250 contiguous nucleotides of any one of SEQ ID NOs: 1-50.

2. The nucleic acid probe of claim 1, wherein the probe comprises:

(a) a nucleic acid molecule having at least 95% sequence identity with the sequence according to any one of SEQ ID NOs: 1-50; or

(b) a nucleic acid molecule having at least 95% sequence identity with at least 250 contiguous nucleotides of any one of SEQ ID NOs: 1-50.

3. The nucleic acid probe of claim 2, wherein the probe comprises:

(a) a nucleic acid molecule having at least 99% sequence identity to the sequence according to any one of SEQ ID NOs; 1-50; or

(b) a nucleic acid molecule having at least 99% sequence identity with at least 250 contiguous nucleotides of any one of SEQ ID NOs: 1-50.

4. The nucleic acid probe of claim 3, wherein the probe comprises:

(a) a nucleic acid molecule consisting of the sequence according to any one of SEQ ID NOs: 1-50; or

(b) a nucleic acid molecule consisting of at least 250 contiguous nucleotides of any one of SEQ ID NOs: 1-50.

5. The nucleic acid probe of claim 1, wherein the nucleic acid probe comprises a nucleic acid molecule with at least 90% sequence identity to at least 400 contiguous of any one of SEQ ID NOs: 1-50.

6. The nucleic acid of claim 5, wherein the nucleic acid probe comprises a nucleic acid molecule with at least 90% sequence identity to at least 500 contiguous nucleotides of any one of SEQ ID NOs: 1-50.

7. The nucleic acid of claim 6, wherein the nucleic acid probe comprises a nucleic acid molecule with at least 90% sequence identity to at least 1000 contiguous nucleotides of any one of SEQ ID NOs: 1-50.

8. The nucleic acid of claim 7, wherein the nucleic acid probe comprises a nucleic acid molecule with at least 90% sequence identity to at least 2500 contiguous nucleotides of any one of SEQ ID NOs: 1-50.

9. An isolated nucleic acid probe comprising two or more portions, wherein:

the first portion comprises at least 250 contiguous nucleotides of a nucleic acid sequence with at least 90% sequence identity to one of SEQ ID NOs: 1-50; and

the second portion comprises at least 250 contiguous nucleotides of a nucleic acid with at least 90% sequence identity to one of SEQ ID NOs: 1-50, wherein the first and second portions are different from one another.

10. The nucleic acid probe of any one of claims 1 to 9, wherein the probe is at least 500 nucleotides in length.

1 1. The nucleic acid probe of claim 10, wherein the probe is at least 1000 nucleotides in length.

12. The nucleic acid probe of claim 11, wherein the probe is at least 5000 nucleotides in length.

13. The nucleic acid probe of any one of claims 1 to 12, further comprising a detectable label.

14. A vector comprising the nucleic acid probe of any one of claims 1 to 13.

15. The vector of claim 14, wherein the vector is a plasmid vector.

16. A probe set comprising at least two of the nucleic acid probes of any one of claims 1 to 13 or at least two of the vectors of claim 14 or claim 15.

17. The probe set of claim 16, wherein the at least two nucleic acid probes are selected from the group consisting of SEQ ID NOs: 1- 10 and a nucleic acid molecule comprising at least 250 contiguous nucleotides of SEQ ID NOs: 1-10.

18. The probe set of claim 17, wherein the at least two nucleic acid probes comprise each of SEQ ID NOs: 1-10.

19. The probe set of claim 16, wherein the at least two nucleic acid probes are selected from the group consisting of SEQ ID NOs: 1 1-20 and a nucleic acid molecule comprising at least 250 contiguous nucleotides of SEQ ID NOs: 1 1 -20.

20. The probe set of claim 19, wherein the at least two nucleic acid probes comprise each of SEQ ID NOs: 1 1-20.

21. The probe set of claim 16, wherein the at least two nucleic acid probes are selected from the group consisting of SEQ ID NOs: 21-30 and a nucleic acid molecule comprising at least 250 contiguous nucleotides of SEQ ID NOs: 21-30.

22. The probe set of claim 21, wherein the at least two nucleic acid probes comprise each of SEQ ID NOs: 21-30.

23. The probe set of claim 16, wherein the at least two nucleic acid probes are selected from the group consisting of SEQ ID NOs: 31-40 and a nucleic acid molecule comprising at least 250 contiguous nucleotides of SEQ ID NOs: 31-40.

24. The probe set of claim 23, wherein the at least two nucleic acid probes comprise each of SEQ ID NOs: 31-40.

25. The probe set of claim 16, wherein the at least two nucleic acid probes are selected from the group consisting of SEQ ID NOs: 41-50 and a nucleic acid molecule comprising at least 250 contiguous nucleotides of SEQ ID NOs: 41-50.

26. The probe set of claim 25, wherein the at least two nucleic acid probes comprise each of SEQ ID NOs: 41-50.

27. A kit comprising one or more of the nucleic acid probes of any one of claims 1 to 13, the vector of claim 14 or claim 15, or one or more probe sets of any one of claims 16 to 26.

28. A method of detecting a target nucleic acid molecule in a sample, comprising: contacting the sample with the nucleic acid probe of any one of claims 1 to 13 or the probe set of any one of claims 16 to 26 under conditions sufficient to permit hybridization between the probe or the probe set and the target nucleic acid; and

detecting the hybridization, wherein presence of hybridization indicates presence of the target nucleic acid in the sample.

29. The method of claim 28, wherein the target nucleic acid is phosphatase and tensin homolog (PTEN) and the probe comprises a nucleic acid sequence having at least 90% sequence identity to one or more of SEQ ID NOs: 1 - 10 or a nucleic acid molecule having at least 90% sequence identity with at least 250 contiguous nucleotides of any one of SEQ

ID NOs: 1-10.

30. The method of claim 28, wherein the target nucleic acid is PTEN and the probe set comprises each of SEQ ID NOs: 1- 10.

31. The method of claim 28, wherein the target nucleic acid is phosphatidylinositol 3- kinase pi 10 (PIK3CA) and the probe comprises a nucleic acid sequence having at least 90% sequence identity to one or more of SEQ ID NOs: 1 1-20 or a nucleic acid molecule having at least 90% sequence identity with at least 250 contiguous nucleotides of any one of SEQ ID NOs: 1 1-20.

32. The method of claim 28, wherein the target nucleic acid is PIK3CA and the probe set comprises each of SEQ ID NOs: 1 1-20.

33. The method of claim 28, wherein the target nucleic acid is mouse double minute 2

(MDM2) and the probe comprises a nucleic acid sequence having at least 90% sequence identity to one or more of SEQ ID NOs: 21-30 or a nucleic acid molecule having at least 90% sequence identity with at least 250 contiguous nucleotides of any one of SEQ ID NOs: 21-30.

34. The method of claim 28, wherein the target nucleic acid is MDM2 and the probe set comprises each of SEQ ID NOs: 21-30.

35. The method of claim 28, wherein the target nucleic acid is Met proto-oncogene (MET) and the probe comprises a nucleic acid sequence having at least 90% sequence identity to one or more of SEQ ID NOs: 31-40 or a nucleic acid molecule having at least 90% sequence identity with at least 250 contiguous nucleotides of any one of SEQ ID NOs: 31-40.

36. The method of claim 28, wherein the target nucleic acid is MET and the probe set comprises each of SEQ ID NOs: 31-40.

37. The method of claim 28, wherein the target nucleic acid is topoisomerase II alpha (TOP2A) and the probe comprises a nucleic acid sequence having at least 90% sequence identity to one or more of SEQ ID NOs: 41-50 or a nucleic acid molecule having at least

90% sequence identity with at least 250 contiguous nucleotides of any one of SEQ ID NOs: 41-50.

38. The method of claim 28, wherein the target nucleic acid is TOP2A and the probe set comprises each of SEQ ID NOs: 41-50.