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Probe design methods and microarrays for comparative genomic hybridization and location analysisRelated Patent Categories: Chemistry: Molecular Biology And Microbiology, Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip, Involving Nucleic AcidProbe design methods and microarrays for comparative genomic hybridization and location analysis description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20060110744, Probe design methods and microarrays for comparative genomic hybridization and location analysis. Brief Patent Description - Full Patent Description - Patent Application Claims
FIELD OF THE INVENTION [0001] The invention relates to methods for designing and selecting probes for microarrays, and in particular comparative genome hybridization arrays and for location analysis. BACKGROUND OF THE INVENTION [0002] Comparative Genomic Hybridization (CGH) and location analysis are important applications, which allow scientists to improve their understanding of the expression and regulation of genes in biological systems. Both CGH and location analysis entail quantifying or measuring changes in copy number of genomic sequences. CGH, is particularly important in developmental biology as well as the causes of cancer and offers great potential in the diagnostics of cancer and developmental diseases. Recently, cDNA microarrays have been used for CGH studies. An oligo-array based approach has several substantial advantages over other technologies, in that it allows the designer to position the probes anywhere within the genomic or polynucleotide sequence of interest. The probes can be placed at whatever density is commensurate with the real-estate or area available on the microarray (in terms of number of features) and the genomic regions of interest can be evaluated by analyzing the hybridization of target sequences to the surface-bound probes. The oligonucleotide probe approach also offers the flexibility of focusing in on regions within exons or introns of expressed sequences, or intergenic regions and regulatory regions for location analysis, as well as any desirable admixture of the aforementioned. [0003] Probes that work well on microarrays for gene expression generally do not work well for CGH arrays and are not appropriate for location analysis arrays. The overall performance of probes for CGH and location analysis arrays entails different optimization of their properties than probes utilized for gene expression. Most notably, these differences relate to the substantially increased complexity of the labeled target mixture for CGH and location analysis than for expression analysis which demands a greater specificity of the probes in discriminating against non-specific binding to competing targets. For comparison, the total number of nucleotide bases in the human transcriptome is approximately 10.sup.8, while the human genome contains over 3.times.10.sup.9 bases. Additionally, probes selected for gene expression come from within message sequences that are transcribed as RNA, i.e. exons, while probes for CGH need be complementary, or nearly so, to contiguous targets selected from within a genome sequence e.g. introns and/or exons. [0004] With increased target complexity comes increased flexibility in the choice of probes. For example, many methods for gene expression restrict probe design to several hundred bases of the 3'-end of the target (message) sequence. Thus, limiting the probe designer to a choice of one in about 500-1000 discrete positions where a probe can be started within any given gene (or transcript). However, for CGH probe design, scientists have a much broader region in which to chose a probe for any given gene. This region may include introns as well as exons and is typically hundreds of thousands of bases long, and in some cases even millions of bases in length. [0005] For location analysis probe design, scientists have a specific region in which to identify and design probes. While the probe designer is constrained to selecting probes within regulatory regions, regions upstream of genes and/or specific locations of interest, the overall number of bases which must be screened is much larger and broader than the region analyzed for gene expression probe design. [0006] Despite great interest in CGH technology, methods for evaluating probes in silico and also empirically for use in this technology are limited. A rigorous method would be to measure signals (e.g. ratios) from each polynucleotide in controlled experiments with test samples containing known copy numbers for each sequence on the array. For example, a method used by several probe designers for measuring array performance for sets of polynucleotides specific for sequences on the X chromosome, is to use a series of cell lines with known variable copies of the X chromosome for CGH experiments. These cell lines (X series) contain intact copies (e.g. 1 to 5) of the X chromosome permitting a rigorous measure of the relationship between copy number and signal intensities for each X chromosome specific polynucleotide on an array. [0007] However, cell lines containing known variable numbers of intact copies of other chromosomes besides for the X chromosome in the genome are not readily available. Furthermore, the aberrant X series cell lines are slow growing and can spontaneously vary in ploidy under standard culturing conditions. Such methods are complex and time-consuming and cannot readily be used to assay the relationship between the hybridization signal of polynucleotides on an array and the genomic copy number of sequences from each chromosome in a cell. [0008] Accordingly, a great need exists for methods for designing and evaluating surface-bound CGH probe nucleic acids (i.e. probes) as well as microarrays comprising these probes which have been identified to have probe properties which make them well suited for CGH and location analysis. This invention meets this, and other, needs. Relevant Literature [0009] United States patents of interest include: U.S. Pat. Nos. 6,465,182; 6,335,167; 6,251,601; 6,210,878; 6,197,501; 6,159,685; 5,965,362; 5,830,645; 5,665,549; [0010] 5,447,841 and 5,348,855. Also of interest are published U.S. application Ser. No. 2002/0006622 and published PCT application WO 99/23256. Articles of interest include: Pollack et al., Proc. Natl. Acad. Sci. (2002) 99: 12963-12968; Wilhelm et al., Cancer Res. (2002) 62: 957-960; Pinkel et al., Nat. Genet. (1998) 20: 207-211; Cai et al., Nat. Biotech. (2002) 20: 393-396; Snijders et al., Nat. Genet. (2001) 29:263-264; Hodgson et al., Nat. Genet. (2001) 29:459-464; Trask, Nat. Rev. Genet. (2002) 3: 769-778; Rabinovitch et al., Cancer Res. (1999) 59:5148-5153; Lee et al., Human Genet. (1997) 100:291:304; Conlon et al. PNAS (2003) 100:3339-3344; Trinklein et al. [0011] Genome Res. (2003) 308-312; J Breslauer et al. Proc Natl Acad Sci. (PNAS) 1986 June; 83(11): 3746-3750; Naoki Sugimoto et al. Nucleic Acids Research, V24, 4505, 1996. SUMMARY OF THE INVENTION [0012] Methods for designing and identifying probes for array based measurements of genomic copy number for comparative genomic hybridization and location analysis are provided. Specifically, a method for generating candidate probes from a target sequence or genomic sequence of interest, repeat-masking the target sequence to form non-repeat masked regions; and tiling, generating a periodic set of sequences across the non-repeat masked regions to generate the candidate probes. [0013] The above method may further comprise screening the candidate probes according to at least one of several in silico parameters and or properties. The method of the invention may also comprise screening the candidate probes according to at least one experimentally measurable parameter or property. The method may further comprise validating the candidate probes by target hybridization experiments. [0014] In some embodiments, the method may further comprise identifying restriction cut sites within the target sequence, and selecting target sequences that exclude or are bounded by these restriction sites when generating candidate probes. Filtering out target sequences with restriction cut sites, reduces the number of possible candidate probes prior to other components of in silico analysis and decreases the amount computational time needed to evaluate the candidate probes. [0015] In other embodiments, the screening according to the in silico parameters comprises annotating the candidate probes for expression and association with the genes of interest. The screening may also comprise analyzing the candidate probes for target specificity and/or thermodynamically annotating the candidate probes. In yet another embodiment, the in silico parameters may comprise a parameter for kinetic properties of the candidate probes. [0016] Methods which comprise in silico annotation may include annotating the candidate probes for their thermodynamic properties, such as duplex melting temperature and/or hairpin stability of the candidate probes. Where the methods of the invention comprise a parameter for duplex melting temperature, the duplex melting temperature may be estimated by the GC-content of the candidate probes. An accurate determination of the melting temperature of oligonucleotide hybridization is achieved by a use of a model that considers nearest-neighbor interactions as represented by the nearest-neighbor parameters. [0017] In other embodiments of the invention, the in silico parameters may comprise a parameter for duplex stability for the candidate probes. In some methods, the duplex stability parameter evaluates the candidate probes for a property selected from the group consisting of melting temperature, entropy, enthalpy and Gibb's free energy. In other embodiments, the hairpin structural stability parameter for the probe, and the target stability parameters may be determined by evaluating the candidate probes for a property selected from the group consisting of melting temperature, entropy, enthalpy and Gibb's free energy. Alternatively, the in silico parameters may be target specificity, and/or target secondary structural stability, where target structural stability is evaluated by a property selected from the group consisting of melting temperature, entropy, enthalpy and Gibb's free energy. [0018] In other methods of the invention, the in silico parameters may comprise a parameter that is the maximum subsequence melting temperature of the probe. [0019] This is the maximum duplex melting for any contiguous sub-sequence of a probe with its complementary target, where all possible subsequences of length L are considered and where L is less than the probe length. This metric has been found to be informative in filtering out probes that have GC-rich regions that appear to act as nucleation sites for non-specific hybridization. For the probes we designed, with nominal lengths of 60 bp, the lengths of L of interest spanned from 15-30 bp. [0020] In other methods of the invention, the in silico parameters may comprise a parameter for intergenicity of the candidate probes. When the parameter for intergenicity is utilized, this parameter evaluates whether the candidate probe sequence is within a gene, in between a gene or within a coding region of a gene. Continue reading about Probe design methods and microarrays for comparative genomic hybridization and location analysis... 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