Patent Application
20050266459
The instant application contains a “lengthy” Sequence Listing which has been submitted via CD-R in lieu of a printed paper copy, and is hereby incorporated by reference in its entirety. Said CD-R, recorded on May 3, 2005, are labeled CRF, “Copy 1” and “Copy 2”, respectively, and each contains only one identical 21.4 MB file (91386us.APP).
The present invention relates to probes for use in various applications. For example, these probes may be used to detect, quantify, identify, or analyze nucleic acid molecules or other molecules that bind to the probes. The invention also encompasses compositions and kits containing the probes and methods of using the probes.
Probe-based assays are useful in the detection, quantitation and analysis of nucleic acids by hybridizing a probe to a nucleic acid sample. Probes have long been used to analyze samples for the presence of nucleic acid from a bacteria, fungi, virus or other organism (See for example; U.S. Pat. Nos. 4,851,330, 5,288,611, 5,567,587, 5,601,984 and 5,612,183). Probe-based assays are also useful in examining genetically-based clinical conditions of interest, as discussed below.
There are several types of probes that may be used for hybridizing to a nucleic acid sample (See generally Szeles, Acta Microbiol. Immunol. Hungarica, 49:69-80 (2002)). These probes include short sequences of genomic DNA or cDNA, whole chromosome paints, chromosome repeats, and whole genomes. In the case of genomic probes, frequently repeated sequences in mammalian genomes have relatively little evolutionary conservation. Thus, total nuclear or genomic DNA can be used as a species-specific probe. Chromosome paints are collections of DNA sequences derived from a single chromosome type and can identify that specific chromosome type in metaphase and interphase nuclei. Different chromosomal types also have unique repeated sequences that may be targeted for probe hybridization (See Cremer et al., Hum. Genet., 74:346-52 (1986)). These chromosomal repeat probes have been cloned for more than two thirds of the human chromosome types (See Szeles, Acta Microbiol. Immunol. Hungarica, 49:69-80 (2002)). Large insert probes that target unique single-copy sequences are another example of a probe type that may be used in hybridization assays. These probes may be in cosmids, bacterial artificial chromosomes (BACs), P1 diverted artificial chromosomes (PACs), or yeast artificial chromosomes (YACs). With these large probes, the hybridization efficiency is inversely proportional to the probe size. Nonetheless, probes as small as 2 kb have been used (See Id.).
Detection of nucleic acid sequences by in situ hybridization, which involves a fundamental process in molecular biology, has been applied for several years (Speel, Histochem. Cell Biol., 112:89-113 (1999)). Sequence differences as subtle as a single base (point mutation) in very short oligomers (<10 base pairs “bp”) can be sufficient to enable the discrimination of the hybridization to complementary nucleic acid target sequences as compared with non-target sequences. Nonetheless, probes of greater than 10 bp in length are generally required to obtain the sequence diversity necessary to correctly identify a unique organism or clinical condition of interest. The ability to discriminate between closely related sequences is inversely proportional to the length of the hybridization probe because the difference in thermal stability decreases between wild type and mutant complexes as the probe length increases. In situ hybridization began by using probes that were radioactively labeled. Currently, however, several alternate detectable labels are available, as discussed below.
Fluorescence in-situ hybridization (“FISH”) is one example of a probe-based assay in which the probes of the invention may be used. FISH was initially developed in the late 1970s (Rudkin and Stollar, Nature, 265:472-73 (1977)). The technique generally entails preparing a cytological sample, labeling probes, denaturing target chromosomes and the probe, hybridizing the probe to the target sequence, and detecting a signal. Typically, the hybridization reaction fluorescently stains the target sequences so that their location, size, or number can be determined using fluorescence microscopy, flow cytometry or other suitable instrumentation. In mammalian cells, chromosomes are arranged in matching pairs. Each chromosome in the pair is a sister chromosome. DNA sequences ranging from whole genomes down to several kilobases can be studied using current hybridization techniques in combination with commercially available instrumentation.
FISH-based staining is sufficiently distinct such that the hybridization signals can be seen both in metaphase spreads and in interphase nuclei. Single and multicolor FISH, using probes, have been applied to different clinical applications generally known as molecular cytogenetics, including prenatal diagnosis, leukemia diagnosis, and tumor cytogenetics. In general, the FISH technique has several advantages including ease of use, rapid results, reduced background in comparison with the radioactive labels that preceded fluorescent labels, and high sensitivity.
The FISH technique has several applications in the clinical setting. These applications include, for example, detection of chromosomal aneuploidy in prenatal diagnoses, hematological cancers, and solid tumors; detection of gene abnormalities such as oncogene amplifications, gene deletions, or gene fusions; chromosomal structural abnormalities such as translocations, duplications, insertions, or inversions; detection of contiguous gene syndromes such as microdeletion syndrome; monitoring the genetic effect of therapy; detection of viral nucleic acids in somatic cells and viral integration sites in chromosomes; gene mapping; and cell cycle analysis (Luke and Shepelsky, Cell Vision 5:49-53 (1998)).
With regard to applications in cancer research, FISH analysis has lead to the identification of several deletion-prone regions in human chromosomes that have been correlated with cancers in various tissues including breast, kidney, lung, uterus, testis, and ovaries (Szeles, Acta Microbiol. Immunol. Hungarica, 49:69-80 (2002)). FISH analysis has also been used to analyze tumor-suppressor genes and DNA repair genes.
There are several variations of the FISH technique those in the art have used to analyze chromosomes (Luke and Shepelsky, Cell Vision 5:49-53 (1998)). For example, in comparative genomic hybridization (CGH) whole genomes are stained and compared to normal reference genomes for the detection of regions with aberrant copy number. FISH is ideally suited for the simultaneous detection of multiple hybridization probes because of the availability of spectrally distinct fluorochromes. In addition, FISH allows the accurate quantitation of hybridization signals. These characteristics paved the way for multicolor applications, which evolved to the simultaneous hybridization of 24 or even more DNA probes for the FISH-based karyotyping chromosomes. For such applications, several techniques, including multiplex FISH (m-FISH; Speicher et al., Nat. Genet., 12:368-75 (1996)), spectral karyotyping (SKY; Schröck et al., Science, 273:494-97 (1996)), combined binary ration labeling (COBRA; Tanke et al., Eur. J. Hum. Genet., 7:2-11 (1999)), or color-changing karyotyping (Henegariu et al., Nat. Genet., 23:263-64 (1999)) have been developed. Moreover, FISH-based banding technologies, such as cross-species color banding (Müller et al., Hum. Genet., 100:271-78 (1997)) or high resolution multicolor banding (Chudoba et al., Cytogenet. Cell Genet., 84:156-60 (1999)) were developed for karyotyping and to facilitate the identification of intrachromosomal rearrangements. Thus, a variety of different multicolor-FISH technologies are available to the skilled artisan. The basic principles of multicolor-FISH were reviewed recently (Lichter, Trends Genet., 13:475-79 (1997)). When used on abnormal material, the probes will stain the aberrant chromosomes thereby deducing the normal chromosomes from which they are derived (Macville M et al., Histochem Cell Biol., 108:299-305 (1997)). Specific DNA sequences, such as the ABL gene, can be reliably stained using probes of only 15 kb (Tkachuk et al., Science, 250: 559-62 (1990)). Telomeric multiplex FISH (TM-FISH) allows for simultaneous hybridization of several subtelomeric probes on one sample slide (Henegariu et al., Lab. Invest., 81:483-91 (2001)). These subtelomeric probes are between 100 kb and 1 Mb from the end of the chromosomes.
SKY uses charge coupled device (CCD) imaging and Fourier spectroscopy to assess the spectrum of fluorescent wavelengths for each pixel scanned, assigning a specific pseudocolor depending on the spectrum identified (Schrock et al. Science, 273:494-497 (1996)). See also the E.C.A. Newsletter for a general description of SKY, available at http://www.biologia.uniba.it/eca/NEWSLETTERS/NS-7/02-Multiplex-FISH.html. In cases of chromosomal aberrations involving small genomic segments, SKY analysis may require supplemental FISH analysis using smaller, subtelomeric probes or chromosome painting probes, (Fan et al., Genetic Testing, 4:9-14 (2000); Hilgenfeld et al., British J. Haematol., 113:305-17 (2001)).
Split-signal FISH (ssFISH) detects changes in chromosomal structure by using two probes, each of which is labeled by a different detectable label. The detectable labels should be distinguishable from one another. Each probe binds to the chromosome on either side of a suspected breakpoint in the chromosome. In a normal chromosome, the two probes will be proximal enough to each other such that the combined signal of their different labels forms a signal that is different from each label alone. Thus, a normal chromosomal sample will contain only the combined or fused signal of the two probes on the sister chromosomes. In an abnormal sample, where one sister chromosome has broken at the suspected break point, the fused signal will remain on the normal sister chromosome. On the broken chromosome, one probe migrates to a completely different location, where the individual signal of that translocated probe becomes apparent. The other individual probe remains on the split chromosome and, because it is no longer proximal to the other probe, emits its individual signal as well. In sum, because of the break in chromosomal structure, the two probes are no longer juxtaposed, allowing the fused signal they cause together to split into the individual signals for each probe. See generally WO 98/51817.
Fusion-signal FISH is similar to ssFISH in that two probes with two different, distinguishable labels are used such that proximity of the two labels produces a new fused signal. The two methods differ in that for fusion-signal FISH, the two probes bind to two different locations that are suspected to become proximal to each other as a result of a chromosomal translocation. Thus, in a normal sample, only signals from the individual probes are present and no fused signal appears. In an abnormal sample, where a piece of one chromosome has attached to another chromosome, the normal chromosomes in each of the two pairs involved will still emit each of the individual probe signals. In the abnormal chromosome, in which the probes are now proximal due to the translocation, the fused signal appears. For a general discussion of fusion-signal FISH, see Arnoldus et al., Cytogenet. Cell Genet., 54:108-11 (1990). Similar results can be obtained without the use of fluorescent microscopy. Such methods include in situ hybridization (ISH) and radioactive in situ hybridization or chromogenic in situ hybridization (CISH). See Tanner et al., Am. J. Pathol., 157:1467-72 (2000).
The invention provides a series of probes that may be used in the above-described assays or in other probe-based assays. For a general discussion of exemplary alternate probe-based assays see Veltman et al., Biotechniques, 35:1066-70 (2003).
The invention relates to probes that may be used in a variety of probe-based assays. In one embodiment, the probes are identical to the nucleic acid sequences set forth in SEQ ID NOS:1-107. In another embodiment, the probes are substantially identical to the nucleic acid sequences set forth in SEQ ID NOS:1-107. In yet another embodiment, the probes are labeled with a detectable label. In another embodiment, the probes are not labeled.
In another embodiment, the probe flanks the breakpoint region of a chromosome. In another embodiment, the probe partially overlaps the breakpoint region of a chromosome. In another embodiment, the probe extends past the breakpoint region of a chromosome.
In yet another embodiment, unlabeled probes are used to remove complementary labeled probes that are at a chromosomal breakpoint, thus producing labeled break point probes. For example, two differentially labeled probes extending into a chromosome breakpoint region may overlap each other. As shown in
In a further embodiment, the nucleic acid probe is selected from (a) a nucleic acid comprising the nucleotide sequence substantially as set out in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ. ID NO. 22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ-ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, or SEQ ID NO:107; and (b) a nucleic acid that hybridizes to the complement of the nucleic acid of (a) under low stringency conditions. In alternate embodiments, hybridization may occur under moderate or high stringency conditions.
In one embodiment, the nucleic acid probe is a fragment of the nucleic acid probes disclosed herein that is complementary to the binding area of interest.
In an additional embodiment, the probes may be used to detect a change in chromosomal structure. These changes in chromosomal structure may be associated with a variety of diseases. For example, lymphomas are frequently associated with translocations. As another example, the human ETV6 gene (Golub et al., Cell, 77:307-16 (1994)), also known as TEL, consists of 8 exons occupying a region of 240 kb on chromosome 12 band p13 (Baens et al., Genome Res., 6:404-13 (1996)). The ETV6 protein contains two critical domains, the 5 helix-loop-helix (HLH) dimerization domain coded by exon 3 and 4, whereas exons 6-8 code a 3′ E26 transformations-specific (ETS) DNA-binding domain (Id.). The ETV6 gene encodes a sequence-specific transcriptional repressor that is required specifically for hematopoiesis within the bone marrow (Wang et al., Genes Dev., 12:2392-402 (1998)). This gene has been identified in fusion with many different partners and in a wide spectrum of human leukemias such as in chronic myelomonocytic leukemia (CMML), acute myeloid leukemia (AML), and acute lymphoblastic leukemia (ALL) (Mitelman et al., available at http://cgap.nci.nih.gov/Chromosomes/Mitelman).
The most common gene rearrangement in cancer of childhood is the fusion of ETV6 and AML1 (also know as RUNX1 and CBFA2) in approximately 22-25% of the cases with childhood B-lineage ALL (Shurtleff et al., Leukemia, 9:1985-89 (1995); Romana et al., Blood, 86:4263-69 (1995)). The ETV6/AML 1 translocation involves the ETV6 disrupting the gene between the HLH motif and the ETS DNA-binding domain. This fusion results in a cryptic t(12;21) translocation that is easily missed by conventional cytogenetic analysis because the rearranged fragments barely affect the morphology of the involved chromosomes (Ferrando and Look, Sem. Hematol., 37:381-95 (2000)). Deletion of the nontranslocated ETV6 allele are frequently associated with a t(12,21) in childhood ALL (Raynaud et al., Blood, 87:2891-99 (1996); Cave et al., Leukemia, 11:1459-64 (1997)). The extent of this deletion is highly variable ranging from, in the majority of cases, where the whole of ETV6 is deleted to a small number where the deletion is intragenic (Raynaud et al., Blood, 87:2891-99 (1996)). The presence of t(12;21) is shown to be a significant favorable prognostic factor, with event-free survivals of 91% in childhood ALL (Rubnitz et al., J. Clin. Oncol., 15:1150-57 (1997)). Table 1 provides a non-exhaustive list of some of the diseases detected by the probes of the invention.
Other embodiments include kits or compositions containing the probes of the invention wherein the kit or composition may be used to diagnose the aforementioned diseases or any other disease also associated with the target sequences bound by the claimed probes.
Additional objects and advantages of the invention will be set forth in part in the description which follows. In addition, the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.
Color versions of
As used herein, the term “probe” refers to a “nucleic acid” probe or to a “nucleic acid analog” probe. As used herein, the term “nucleic acid” refers to a nucleobase sequence-containing oligomer, polymer, or polymer segment, having a backbone formed solely from naturally occurring nucleotides or unmodified nucleotides. As used herein, a “nucleic acid analog” means an oligomer, polymer, or polymer segment composed of at least one modified nucleotide, or subunits derived directly from a modification of nucleotides. Non-limiting examples of naturally occurring nucleobases include: adenine, cytosine, guanine, thymine, and uracil. Non-limiting examples of modified nucleotides include: 5-propynyl-uracil, 2-thio-5-propynyl-uracil, 5-methylcytosine, pseudoisocytosine, 2-thiouracil and 2-thiothymine, 2-aminopurine, N9-(2-amino-6-chloropurine), N9-(2,6-diaminopurine), hypoxanthine, N9-(7-deaza-guanine), N9-(7-deaza-8-aza-guanine) and N8-(7-deaza-8-aza-adenine). Other non-limiting examples of suitable nucleobases include those nucleobases illustrated in FIGS. 2(A) and 2(B) of Buchardt et al. (U.S. Pat. No. 6,357,163).
The term “nucleic acid analog” also refers to synthetic molecules that can bind to a nucleic acid. For example, a nucleic acid analog probe may be comprised of peptide nucleic acids (PNAs), locked nucleic acids (LNAs), or any derivatized form of a nucleic acid. As used herein, the term “peptide nucleic acid” or “PNA” means any oligomer or polymer comprising at least one or more PNA subunits (residues), including, but not limited to, any of the oligomer or polymer segments referred to or claimed as peptide nucleic acids in U.S. Pat. Nos. 5,539,082, 5,527,675, 5,623,049, 5,714,331, 5,718,262, 5,736,336, 5,773,571, 5,766,855, 5,786,461, 5,837,459, 5,891,625, 5,972,610, 5,986,053, 6,107,470 6,201,103, 6,228,982 and 6,357,163; all of which are herein incorporated by reference. The term PNA also applies to any oligomer or polymer segment comprising two or more subunits of those nucleic acid mimics described in the following publications: Lagriffoul et al., Bioorganic & Medicinal Chemistry Letters, 4:1081-1082 (1994); Petersen et al., Bioorganic & Medicinal Chemistry Letters, 6: 793-796 (1996); Diderichsen et al., Tett. Lett. 37: 475-478 (1996); Fujii et al., Bioorg. Med. Chem. Lett. 7: 637-627 (1997); Jordan et al., Bioorg. Med. Chem. Lett. 7: 687-690 (1997); Krotz et al., Tett. Lett. 36: 6941-6944 (1995); Lagriffoul et al., Bioorg. Med. Chem. Lett. 4: 1081-1082 (1994); Diederichsen, U., Bioorganic & Medicinal Chemistry Letters, 7:1743-1746 (1997); Lowe et al., J. Chem. Soc. Perkin Trans. 1, (1997) 1: 539-546; Lowe et al., J. Chem. Soc. Perkin Trans. 11: 547-554 (1997); Lowe et al., J. Chem. Soc. Perkin Trans. 11:555-560 (1997); Howarth et al., J. Org. Chem. 62:5441-5450 (1997); Altmann, K-H et al., Bioorganic & Medicinal Chemistry Letters, 7:1119-1122 (1997); Diederichsen, U., Bioorganic & Med. Chem. Lett., 8:165-168 (1998); Diederichsen et al., Angew. Chem. Int. Ed., 37: 302-305 (1998); Cantin et al., Tett. Lett., 38:4211-4214 (1997); Ciapetti et al., Tetrahedron, 53: 1167-1176 (1997); Lagriffoule et al., Chem. Eur. J., 3:912-919 (1997); Kumar et al., Organic Letters 3(9): 1.269-1272 (2001); and the Peptide-Based Nucleic Acid Mimics (PENAMs) of Shah et al. as disclosed in WO96/04000. As used herein, the term “locked nucleic acid” or “LNA” means an oligomer or polymer comprising at least one or more LNA subunits. As used herein, the term “LNA subunit” means a ribonucleotide containing a methylene bridge that connects the 2′-oxygen of the ribose with the 4′-carbon. See generally, Kurreck, Eur. J. Biochem., 270:1628-44 (2003).
The term “identical” means that a relevant sequence is 100% identical to the non-repetitive region of a given sequence. The term “substantially identical,” or “substantially” means that a relevant sequence is at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to the non-repetitive region of a given sequence. By way of example, such sequences may be allelic variants, sequences derived from various species, or they may be derived from the given sequence by truncation, deletion, amino acid substitution or addition. For nucleic acids, the length of comparison sequences will generally be at least 50, 100, 150, 300, or more nucleotides. Percent identity between two sequences is determined by standard alignment algorithms such as, for example, Basic Local Alignment Tool (BLAST) described in Altschul et al., J. Mol. Biol., 215:403-410 (1990), the algorithm of Needleman et al., J. Mol. Biol., 48:444-453 (1970), or the algorithm of Meyers et al., Comput. Appl. Biosci., 4:11-17 (1988).
In one embodiment, the nucleic acid probe is selected from (a) a nucleic acid comprising at least one nucleotide sequence substantially as set out in Table 2; and (b) a nucleic acid that hybridizes to the complement of the nucleic acid of (a) under low stringency conditions.
In an alternate embodiment, the nucleic acid probes of the invention are identical to at least one of the sequences identified in Table 2.
A probe binds to a target sequence under certain conditions. The term “bind” is synonymous with “hybridize.” When two molecules hybridize, they form a combination of the two molecules through one or more types of chemical bonds, through complementary base pairing, or through hydrogen bond formation. In addition, bases in a probe may be joined by a linkage other than a phosphodiester bond, so long as it does not prevent hybridization. Thus, oligonucleotide probes may have constituent bases joined by peptide bonds rather than phosphodiester linkages. As used herein, the term complementary refers to nucleobases that may hybridize to each other. For example, adenine is complementary to thymine and cytosine is complementary to guanine. As used herein, the term “binds under certain conditions” is intended to describe conditions for hybridization and washes under which nucleotide sequences that are significantly identical or homologous to each other remain bound to each other. The conditions may be such that sequences, which are at least about 70%, such as at least about 80%, and such as at least about 85-90% identical, remain bound to each other. As used herein, the term “target sequence” refers to the nucleobase sequence sought to be determined. The nucleobase sequence can be a subsequence of a nucleic acid molecule of interest (e.g. a chromosome). In other embodiments, the target sequences are located in the ETV6 gene, the MLL gene, the TCF3 gene (also known as the E2A gene), or the TLX3 gene (also known as the HOX11L2 gene).
Appropriate hybridization conditions can be selected by those skilled in the art with minimal experimentation as exemplified in Ausubel et al. (1995), Current Protocols in Molecular Biology, John Wiley & Sons, sections 2, 4, and 6. Additionally, stringency conditions are described in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Press, chapters 7, 9, and 11. As used herein, defined conditions of “low stringency” are as follows. Filters containing DNA are pretreated for 6 h at 40° C. in a solution containing 35% formamide, 5×SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500 μg/ml denatured salmon sperm DNA. Hybridizations are carried out in the same solution with the following modifications: 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 μg/ml salmon sperm DNA, 10% (wt/vol) dextran sulfate, and 5-20×106 cpm of 32P-labeled probe is used. Filters are incubated in hybridization mixture for 18-20 h at 40° C., and then washed for 1.5 h at 55° C. In a solution containing 2×SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS. The wash solution is replaced with fresh solution and incubated an additional 1.5 h at 60° C. Filters are blotted dry and exposed for autoradiography.
As used herein, defined conditions of “moderate stringency” are as follows. Filters containing DNA are pretreated for 7 h at 50° C. in a solution containing 35% formamide, 5×SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500 μg/ml denatured salmon sperm DNA. Hybridizations are carried out in the same solution with the following modifications: 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 μg/ml salmon sperm DNA, 10% (wt/vol) dextran sulfate, and 5-20×106 cpm of 32P-labeled probe is used. Filters are incubated in hybridization mixture for 30 h at 50° C., and then washed for 1.5 h at 55° C. In a solution containing 2×SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS. The wash solution is replaced with fresh solution and incubated an additional 1.5 h at 60° C. Filters are blotted dry and exposed for autoradiography.
As used herein, defined conditions of “high stringency” are as follows. Prehybridization of filters containing DNA is carried out for 8 h to overnight at 65° C. in buffer composed of 6×SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 μg/ml denatured salmon sperm DNA. Filters are hybridized for 48 h at 65° C. in the prehybridization mixture containing 100 μg/ml denatured salmon sperm DNA and 5-20×106 cpm of 32P-labeled probe. Washing of filters is done at 37° C. for 1 h in a solution containing 2×SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA. This is followed by a wash in 0.1×SSC at 50° C. for 45 minutes.
Other conditions of low, moderate, and high stringency well known in the art (e.g., as employed for cross-species hybridizations) may be used if the above conditions are inappropriate (e.g., as employed for cross-species hybridizations).
In one embodiment, the probes comprise a detectable label. As used herein, a detectable label refers to moieties that can be attached directly or indirectly to an oligomer or polymer to thereby render the oligomer or polymer detectable by an instrument or method. In one embodiment, a detectable label may be directly attached to a probe. In another embodiment, a detectable label may be indirectly attached to a probe. For example, a detectable label may be attached to a probe by using a linker. A detectable label may be, for example, a fluorochrome, a chromophore, a spin label, a radioisotope, an enzyme, a hapten, Quantum Dot, beads, aminohexyl, pyrene, and a chemiluminescence compound, such as acridinione. Fluorochromes that may be used in the method of the present invention include, but are not limited to, IR dyes, Dyomics dyes, phycoerythrine, cascade blue, Oregon green 488, pacific blue, rhodamine green, 5(6)-carboxyfluorescein, cyanine dyes (i.e., Cy2, Cy3, Cy 3.5, Cy5, Cy5.5, Cy 7) (diethyl-amino)coumarin, fluorescein (i.e., FITC), tetramethylrhodamine, lissamine, Texas Red, AMCA, TRITC, and Alexa dyes. Haptens that may be used in the present invention include, but are not limited to, 5(6)-carboxyfluorescein, 2,4-dinitrophenyl, digoxigenin, rhodamine, bromodeoxy uridine, acetylaminoflurene, mercury trinitrophenol, estradiol, and biotin. Enzymes that may be used in the present invention include, but are not limited to, soybean peroxidase, alkaline phosphatase, and horseradish peroxidase.
The probes of the invention may constitute part of a kit. In one embodiment, the kit is comprised of (a) at least two of the probes of the invention and (b) other reagents and compositions for performing an assay to detect a change in chromosomal structure. The invention also encompasses methods for using the probes of the invention. For example, in methods for detecting a change in chromosomal structure. In another embodiment, the method comprises (a) obtaining a preparation of chromosomes; (b)contacting the preparation of chromosomes with at least two of the probes of the invention, wherein the probes each contain a different label distinguishable from the other label and wherein the labeled probes create a pattern of staining in a sample of normal control chromosomes; and (c) detecting the change in chromosomal structure by detecting a change in the pattern of staining in the chromosome preparation as compared to the normal control sample.
As used herein, the term “change in chromosomal structure” refers to an alteration in the chromosomes in a test sample as compared to a normal chromosome control. As used herein, a euploidy is the condition of having a normal number of structurally normal chromosomes. For example, somatic euploid cells from a human female contain 44 autosomal chromosomes and two X-chromosomes for a total of 46 chromosomes or 23 chromosomal pairs. Euploid bulls have 58 autosomal chromosomes, one X-chromosome, and one Y-chromosome.
Non-exhaustive examples of chromosomal alterations include aneuploidy, gene amplifications, deletions including gene deletions, gene fusions, translocations, duplications, insertions, or inversions. As used herein, aneuploidy refers to any deviation from the normal euploid state or the condition of having less than or more than the normal diploid number of chromosomes. Aneuploidy is the most frequently observed type of cytogenetic abnormality. Generally, aneuploidy is recognized as a small deviation from euploidy because major deviations are rarely detected because of the lethal nature of major changes in chromosome number. As used herein, an amplification refers to an increase in the number of copies of a specific DNA fragment. Such DNA fragments include, for example, a gene or an entire chromosome. As used herein, a deletion refers to a genetic event in which a nucleic acid sequence has been removed from a chromosome. As used herein, a gene fusion refers to an accidental joining of the DNA of two genes. Gene fusions may occur by translocations or inversions. Gene fusions may give rise to hybrid proteins or the misregulation of the transcription of one gene due to the juxtaposition of cis regulatory elements (e.g., enhancers or promoters) of another gene. As used herein, a translocation refers to a genetic event in which a part of the nucleic acid sequence of one chromosome is removed from that chromosome and attached to a different chromosome. As used herein, a duplication refers to the repetition of a nucleotide sequence in a chromosome or a chromosome segment. For example, a duplication may result in the repetition of a nucleotide sequence in linear juxtaposition to the duplicated sequence. As used herein, an insertion refers to a genetic event in which a nucleic acid sequence has been introduced between two points in a chromosome. As used herein, an inversion is a genetic event in which a nucleic acid sequence's orientation in a chromosome has been reversed. As used herein, a chromosomal breakpoint refers to a location in the chromosome where the chromosome breaks into two pieces.
As used herein, a preparation of chromosomes refers to a composition comprised of chromosomes from a cell type of interest. A cell type of interest may be a mammalian cell. In a further embodiment, chromosome preparations may be produced from mammalian cells in various stages of mitosis. For example, chromosomal preparations may be produced from cells in metaphase. FISH may also be used on a preparation of chromosomes from cells in interphase. Methods for producing a chromosomal preparation and nuclei for use in FISH analysis are well known to those of skill in the art.
As used herein, the phrases “chosen from one or more of,” “chosen from at least one of,” “chosen from one or more,” and “chosen from at least one” followed by a list of items such as A, B, and C, indicate that one or more of A, B, and C may be selected (e.g. only A; only B; only C; only A and B; only B and C; only A and C; or A, B, and C), and also indicate that one or more types within each A, B, or C category may be selected (e.g. only A1 and A2; only A1 and A2 and B1; only B1 and B2 and C1, etc.).
Reference will now be made in detail to the following Examples, which are provided solely to further describe the invention. These Examples are in no way intended to limit the scope or meaning of the claims.
Unique sequence probes were prepared as follows. Cells containing vectors comprising the probe of interest were grown in appropriate conditions for each cell line. DNA containing the probe sequence was isolated from the cell culture. See generally Zhao and Stodolsky, Methods in Molecular Biology: Library Construction, Physical Mapping, and Sequencing, Vol. 1, 1st ed., Humana Press (2004).
The probe precursor DNA was directly labeled with a fluorochrome by nick translation. On ice, 10 μg of DNA was resuspended in 250 μl of reaction buffer. The reaction buffer contained 25 μl 10× nick translation buffer (500 mmol/L Tris-HCl, 100 mmol/L MgCl2, 1 mmol/L DTT, 100 mg/L BSA, pH 7.5); 25 μl 10×dNTP mix (50 mmol/L Tris-HCl, 10 mmol/L EDTA, 0.5 mmol/L dATP, 0.5 mmol/L dGTP, 0.5 mmol/L dCTP, 0.34 mmol/L dTTP, pH 7.6); 4 μl of dUTP fluorochrome (1 mmol/L); 10 μl DNA polymerase I (Invitrogen, 10 μl); and 0.3 μl DNase I (Sigma Aldrich). The labeling reaction was incubated for 4 hours at 15° C. The reaction was stopped by adding 25 μl 500 mmol/L EDTA and incubating for 10 minutes at 65° C. Unincorporated nucleotides were removed by centrifugation using Microcon YM-10 Centrifugal Filter Devices (Milipore). The purified labeled probe was resuspended in 25 μl of ice cold (−20° C.)TE buffer (10 mmol/L Tris HCl, 0.1 mmol/L EDTA, pH 8.0).
A slide containing metaphase spreads and interphase nuclei was pretreated in TBS (50 mmol/L Tris, 150 mmol/L NaCl, pH 7.6) with 3.7% formaldehyde for 2 minutes at room temperature, as described in Human Cytogenetics, a Practical Approach, Volume 1, 2nd ed., D. E. Rooney and B. H. Czepulkowski editors (1992). The slide was then rinsed twice in phosphate buffered saline (PBS) for 5 minutes per wash at room temperature. After rinsing, the slide was dehydrated in a cold (4° C.) series of ethanol by incubating the slide in 70% ethanol at 4° C. for 2 minutes; incubating the slide in 85% ethanol at 4° C. for 2 minutes; and then incubating the slide in 96% ethanol at 4° C. for 2 minutes, followed by air drying.
On each target area, 10 μL of DNA Hybridization Buffer (100 ng fluorescein labelled probe, 100 ng Texas Red labelled probe, 5 μM PNA Oligo Mix, 45% formamide, 300 mM NaCl, 5 mM NaPO4, 10% Dextran sulphate) was added and an 18×18 mm coverslip was applied to cover the hybridization area. The edges of the coverslip were sealed with rubber cement before denaturation at 82° C. for 5 minutes. The slide was hybridized overnight at 45° C. After hybridization, the coverslip was removed and the slide was washed for 10 minutes in a stringent wash buffer (0.2×SSC, 0.1% Triton X-100) at 65° C.
The slide was then rinsed in TBS for 1 minute before dehydrated in a cold (4° C.) series of 70%, 85% and 96% ethanol as described above. Each slide was mounted with 10 μL of anti-fade solution (Vectashield H-1000, Vector Laboratories, Inc. Burlingame) supplemented with 0.1 μg/mL 4,6-diamidino-2-phenylindole (DAPI, Sigma Chemicals) and sealed with a coverslip. The slide was analyzed using a fluorescence microscope equipped with a CCD digital camera.
BCL2 probes were hybridized to metaphase chromosomes from metaphase spreads from normal peripheral blood. Two normal BCL2 loci on chromosomes 18 were represented by two yellow dots or co-localized green/red signals.
BCL3 probes were hybridized to metaphase chromosomes from metaphase spreads from normal peripheral blood. Two normal BCL3 loci on chromosomes 19 were represented by two yellow dots or co-localized green/red signals.
BCL6 probes were hybridized to metaphase chromosomes from metaphase spreads from normal peripheral blood. Two normal BCL6 loci on chromosomes 3 were represented by two yellow dots or co-localized green/red signals.
CCND1 probes were hybridized to metaphase chromosomes from metaphase spreads from normal peripheral blood. Two normal CCND1 loci on chromosomes 11 were represented by two yellow dots or co-localized green/red signals.
MYC probes were hybridized to metaphase chromosomes from metaphase spreads from normal peripheral blood. Two normal MYC loci on chromosomes 8 were represented by two yellow dots or co-localized green/red signals.
MALT probes were hybridized to metaphase chromosomes from metaphase spreads from normal-peripheral blood. Two normal MALT loci on chromosomes 18 were represented by two yellow dots or co-localized green/red signals.
PAX5 probes were hybridized to metaphase chromosomes from metaphase spreads from normal peripheral blood. Two normal PAX5 loci on chromosomes 9 were represented by two yellow dots or co-localized green/red signals.
TLX1 probes were hybridized to metaphase chromosomes from metaphase spreads from normal peripheral blood. Two normal TLX1 loci on chromosomes 10 were represented by two yellow dots or co-localized green/red signals.
PDGFRA probes were hybridized to metaphase chromosomes of metaphase spreads and nuclei from the cell line ELO-1, and to nuclei from normal tissue. On the metaphase spreads, one normal PDGFRA locus on chromosome 4 was represented by a yellow dot or co-locolized green/red signals. Lack of one red signal indicated a sub-deletion. An additional chromosome 4 could be detected as a result of an amplification of the derivative chromosome 4. On the nuclei, one normal PDGFRA locus on chromosome 4 was represented by a yellow dot or co-locolized green/red signals. Lack of one red signal indicated an sub-deletion. An additional chromosome 4 could be detected as a result of an amplification of the derivative chromosome 4.
PDGFRB probes were also hybridized to metaphase chromosomes from metaphase spreads from the cell line NALM-6. One normal PDGFRA locus on chromosome 5 was represented by a yellow dot or co-locolized green/red signals. Lack of one green signal indicated an deletion of the derivative chromosome 5, while a red signal indicated a translocation to another chromsome.
Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only. All references either discussed or referred to herein are incorporated by reference.
This application claims priority to U.S. Provisional Patent Application Nos. 60/567,570 and 60/567,440, both of which were filed May 4, 2004, and which are incorporated herein by reference in their entirety.
| Number | Date | Country | |
|---|---|---|---|
| 60567570 | May 2004 | US | |
| 60567440 | May 2004 | US |
