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Iterative probe design and detailed expression profiling with flexible in-situ synthesis arraysRelated 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 AcidIterative probe design and detailed expression profiling with flexible in-situ synthesis arrays description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20060141501, Iterative probe design and detailed expression profiling with flexible in-situ synthesis arrays. Brief Patent Description - Full Patent Description - Patent Application Claims
[0001] This is a divisional of copending U.S. patent application Ser. No. 09/561,487, filed on Apr. 28, 2000, which is a continuation-in-part application of U.S. patent application Ser. No. 09/364,751, filed on Jul. 30, 1999, now abandoned, which claims benefit under 35 U.S.C. .sctn. 119(e) of U.S. Provisional Patent Application Ser. No. 60/144,382, filed on Jul. 16, 1999, each of which is incorporated herein by reference in its entirety. 1. FIELD OF THE INVENTION [0002] The field of this invention relates to materials and methods to detect and report polynucleotide sequences, including genomic sequences, genomic transcript sequences (e.g., mRNAs from cells and/or cDNA sequences derived therefrom), copy numbers and SNPs. In particular, the invention relates to methods for detecting polynucleotide sequences using sets of polynucleotide probes that have been selected for optimum sensitivity and specificity. The invention also relates to methods for selecting sets of polynucleotide probes for optimum sensitivity and specificity which may be used, e.g., to detect and report gene expression changes in a cell or cells. The invention further relates to sets of polynucleotide probes, including microarrays comprising such sets of polynucleotide probes, which are selected for optimum sensitivity and specificity and are therefore useful, e.g., to detect and report gene expression changes in a cell or cells. 2. BACKGROUND [0003] Within the past decade, several technologies have made it possible to monitor the expression level of a large number of genetic transcripts at any one time (see, e.g., Schena et al., 1995, Science 270:467-470; Lockhart et al., 1996, Nature Biotechnology 14:1675-1680; Blanchard et al., 1996, Nature Biotechnology 14:1649; Ashby et al., U.S. Pat. No. 5,569,588, issued Oct. 29, 1996). For example, techniques are known for preparing microarrys of cDNA transcripts (see, e.g., DeRisi et al., 1996, Nature Genetics 14:457-460; Shalon et al., 1996, Genome Res. 6:689-645; and Schena et al., 1995, Proc. Natl. Acad. Sci. U.S.A. 93:10539-11286). Alternatively, high-density arrays containing thousand of oligonucleotides complementary to defined sequences, at defined locations on a surface using photolithographic techniques for synthesis in situ are described, e.g., Fodor et al., 1991, Science 251:767-773; Pease et al., 1994, Proc. Natl. Acad. Sci. U.S.A. 91:5022-5026; Lockhart et al., 1996, Nature Biotechnology 14:1675; U.S. Pat. Nos. 5,578,832; 5,556,752;l and 5,510,270). Methods for generating arrays using inkjet technology for oligonucleotide synthesis are also known in the art (see, e.g., Blanchard, International Patent Publication WO 98/41531, published Sep. 24, 1998; Blanchard et al., 1996, Biosensors and Bioelectronics 11:687-690; Blanchard, 1998, in Synthetic DNA Arrays in Genetic Engineering, Vol. 20, J. K. Setlow, Ed., Plenum Press, New York at pages 111-123). [0004] Applications of this technology include, for example, identification of genes which are up regulated or down regulated in various physiological states, particularly diseased states. Additional exemplary uses for transcript arrays include the analyses of members of signaling pathways, and the identification of targets for various drugs. See, e.g., Friend and Hartwell, International Publication No. WO 98/38329 (published Sep. 3, 1998); Stoughton, U.S. Pat. No. 6,132,969; Stoughton and Friend, U.S. Pat. No. 5,965,352; Friend and Stoughton, U.S. Provisional Application Ser. Nos. 60/084,742 (filed May 8, 1998), 60/090,004 (filed Jun. 19, 1998), and 60/090,046 (filed Jun. 19, 1998). [0005] However, several factors limit the number of genetic transcripts that can be detected on a single microarray "chip." In particular, the "reporting density" (i.e., the number of genes detected per unit of surface area) for a microarray is limited, e.g., by the density with which polynucleotide probes may be laid down as well as by the number of polynucleotide probes required per gene. A plurality of probe pairs, which are both matched to and intentionally mismatched to a target sequence, are required in order to empirically distinguish signal arising from a target polynucleotide sequence of interest (e.g., a particular mRNA sequence of interest) from signal arising from cross-hybridization with other polynucleotide sequences. Currently, in situ synthesized microarray chips require more than 20 oligonucleotide probe pairs per gene or gene region reported (Lockhart et al.,supra). On the other hand, the number of polynucleotide probes that may be laid down on a microarray chip is limited by the technology used to produce the microarray. Photolithographic techniques discussed above for producing oligonucleotide microarrays having a high spatial density of probes are expensive to synthesize and therefore require a large capital investment. Oligonucleotide microarrays produced using the above discussed inkjet technology methods are, by contrast, much cheaper and faster to produce both per chip design and per chip. Thus, such microarrays are generally preferred for detecting genetic transcripts in cells. However, microarray chips produced by such inkjet technology have a limited probe density that is only a fraction of the probe density of chips produced by photolithography methods. Thus, at present the number of genetic transcripts that may be detected on a single microarray chip is limited to about 10,000 gene transcripts using expensive, photolithographic arrays, and only about 750 to 2,500 gene transcripts using less expensive, inkjet arrays. [0006] There exists therefore a need for materials and methods which may be used to efficiently detect large numbers of different genetic transcripts and thereby detect changes in a large number of genetic transcripts in a cell or cells. In particular, there is a need for materials and methods which may be used to detect changes in genetic transcription across the entire genome of a cell, including cells of complex organisms such as mammalian cells and, in particular, human cells. [0007] There also exists, however, a need for materials and methods which may be used to accurately detect changes in genetic transcripts in cells, e.g., in response to some environmental change or perturbation. In particular, there is a need to accurately detect changes in the expression levels of those particular genetic transcripts that exhibit the largest changes, e.g., in response to an environmental change or perturbation, and which are therefore most relevant in understanding the effect of the environmental change or perturbation on the cell or cells. [0008] Discussion or citation of a reference herein shall not be construed as an admission that such reference is prior art to the present invention. 3. SUMMARY OF THE INVENTION [0009] The present invention provides methods and compositions that efficiently detect and accurately report gene expression changes in an organism. In particular, the methods and compositions of the invention may be used to detect and report gene expression changes in a cell or organism that occur, e.g., in response to some change or "perturbation" to the cell or organism and/or to its environment, such as exposure of the cell or organism to one or more drugs. [0010] The compositions and methods of the invention use "screening chips," which may be used, e.g., to detect changes in gene expression among a large number of genes or gene transcripts. For example, in particularly preferred embodiments, the screening chips may be used to detect changes in gene expression in the entire genome of an organism. Such screening chips are therefore provided as part of the present invention, as well as methods for making and using such screening chips, e.g., to screen the entire genome of an organism for changes in response to one or more perturbations. [0011] The compositions and methods of the invention also provide "signature chips" which may be used to accurately detect changes in gene expression in a smaller number of genes. For example, the signature chips of the invention may be used to accurately detect changes in the expression of certain "signature genes." In preferred embodiments, the signature genes are those genes whose expression changes the most in response to a particular perturbation or in response to a particular type or set of perturbations (e.g., responses to several doses of a drug or responses to several different, but related drugs). For example, signature genes may be identified using the screening chips of the invention to identify those genes whose expression changes the most in response to a particular perturbation or perturbations. In one preferred embodiment, the signature chips of the invention comprise at least a first probe and a second probe for each signature gene to be detected, wherein the first probe for a particular signature gene is a matched probe having a polynucleotide sequence that is complementary to the particular signature gene or to a portion thereof, and wherein the second probe for a particular signature gene is a mismatch probe having a polynucleotide sequence that is a variant of a sequence which is complementary to the particular signature gene. In another preferred embodiment the signature chips of the invention comprise a plurality of matched probes for each signature gene to be detected, wherein each matched probe for a particular signature gene has a polynucleotide sequence that is complementary to the particular signature gene or to a portion thereof. [0012] The invention also provides methods and compositions for ranking and/or selecting probes according to other parameters including, but not limited to: (a) probe size or length; (b) binding energies, including both the perfect match duplex (i.e., of a probe and its target, complementary nucleotide sequence) and cross-hybridization binding energies; (c) base composition, including, for example, the relative amount or percentage of one or more particular nucleotide bases (e.g., adenine, guanine, thymine or cytosine) in a probe sequence, as well as the relative amount or percentage of any combination of such nucleotide bases; (d) the position of a probe's complementary sequence in the sequence of its "target" polynucleotide or gene sequence; and (e) probe sequence complexity, including the presence or lack of common repetitive elements such as polynucleotide repeats (i.e., simple, contiguous repeats of one or more nucleotide bases) as well as more complicated repetitive elements that are well known in the art. Still other exemplary parameters which can be used in the methods and compositions of the invention for ranking and/or selecting oligonucleotide probes include: (f) self dimer binding energy (i.e., the tendency for a particular probe to hybridize to its own sequence); (g) the structure content of the complementary, target polynucleotide sequence for a particular probe (e.g., the presence or absence of certain structural features or motifs); and (h) the information content of a probe's nucleotide sequence. [0013] The invention is based, at least in part, on the discovery that the number of probe sequences required to reliably and accurately report a particular polynucleotide sequence, such as the sequence of a particular gene, may be reduced to as few as one probe by carefully selecting probes according to the methods and/or having the particular lengths disclosed herein. Accordingly, the invention also provides methods by which probes (i.e., probe sequences) may be ranked and/or selected according to their reporting properties, including, for example, their specificity and sensitivity for a particular sequence (e.g., for the sequence of a particular gene or gene transcript). [0014] The invention thus provides methods for selecting one or more different polynucleotide probes from a plurality of polynucleotide probes according to the sensitivity and specificity with which each different polynucleotide probe hybridizes to a target polynucleotide. In one embodiment, the methods comprise: (a) identifying polynucleotide probes in the plurality of different polynucleotide probes that hybridize to the target polynucleotide with a sensitivity above a threshold sensitivity level; (b) ranking the identified polynucleotide probes according to the specificity with which each identified polynucleotide probe hybridizes to the target polynucleotide; and (c) selecting one or more different polynucleotide probes from the ranked polynucleotide probes. In another embodiment, the methods comprise: (a) identifying polynucleotide probes in the plurality of different polynucleotide probes that hybridize to the target polynucleotide with a specificity above a threshold specificity level; (b) ranking the identified polynucleotide probes according to the sensitivity with which each identified polynucleotide probe hybridizes to the target polynucleotide; and (c) selecting one or more different polynucleotide probes from the ranked polynucleotide probes. In still another embodiment, the methods comprise: (a) ranking the plurality of different polynucleotide probes according to the sensitivity with which each polynucleotide probe hybridizes to the target polynucleotide so that a sensitivity rank is obtained for each different polynucleotide probe; (b) ranking the plurality of different polynucleotide probes according to the specificity with which each polynucleotide probe hybridizes to the target polynucleotide so that a specificity rank is obtained for each different polynucleotide probe; (c) obtaining a combined rank for each different polynucleotide probe, wherein the combined rank is determined by determining the sum of the sensitivity rank and the specificity rank for each different polynucleotide probe; and (d) selecting one or more different polynucleotide probes from the plurality of different polynucleotide probes according to the combined rank of the different polynucleotide probes. In one aspect of this particular embodiment, the sum of the sensitivity rank and the specificity rank for each different polynucleotide probe can be, e.g., a weighted sum of the sensitivity rank and the specificity rank for each different polynucleotide probe. [0015] The invention provides numerous different aspects of these different embodiments for example, the invention provides aspects of the above embodiments wherein the sensitivity with which a particular polynucleotide probe hybridizes to the target is provided by determining the binding energy with which the target polynucleotide hybridizes to the particular polynucleotide probe, e.g., according to the nearest neighbor model. The invention also provides aspects of the above embodiments wherein the sensitivity with which a particular polynucleotide probe hybridizes to the target polynucleotide is provided by a method comprising determining the level of hybridization of the target polynucleotide sequence to the particular polynucleotide probe; e.g., by calculating the level of hybridization of the target polynucleotide to the polynucleotide probe from the binding energy with which the target polynucleotide hybridizes to the particular polynucleotide probe. [0016] In another aspect of the methods of the invention, the specificity with which a particular polynucleotide probe hybridizes to the target polynucleotide is provided, e.g., by: (a) determining the level of hybridization of the target polynucleotide to the particular polynucleotide probe; and (b) determining the level of cross-hybridization of non-target polynucleotides to the particular probe. [0017] In still other embodiments, the methods of the invention comprise: (a) hybridizing a reference polynucleotide sample comprising molecules of the target polynucleotide to the plurality of different polynucleotide probes under conditions such that the hybridization intensity of each different polynucleotide probe to the reference sample correlates with the sensitivity and specificity with which the each different polynucleotide probe hybridizes to the target polynucleotide; and (b) selecting polynucleotide probes in the plurality of different polynucleotide probes that have the highest hybridization intensity. For example, the invention provides particular aspects of this embodiment wherein the hybridization is within 5.degree. C. or within 2.degree. C. of the mean melting temperature of the plurality of different polynucleotide probes from the target polynucleotide. [0018] The invention also provides a preferred embodiment wherein the specificity of a particular polynucleotide probe is provided by a method which comprises selecting, from a plurality of binding energies, a binding energy that indicates the specificity of the particular polynucleotide probe. Specifically, in such a preferred embodiment, the provided plurality of binding energies are binding energies for hybridization of the particular polynucleotide probe to each of a plurality of different polynucleotides, wherein each polynucleotide in the plurality of different polynucleotides is different from the target polynucleotide. The selected binding energy is the largest binding energy in the plurality of binding energies. [0019] For example, in one aspect of this preferred embodiment, the binding energies provided for hybridization of the particular polynucleotide probe to each of the plurality of polynucleotides is provided according to a nearest neighbor model. In one aspect the plurality of polynucleotides comprise polynucleotides expressed by a cell or organism of interest. In one aspect, the plurality of polynucleotides consists of polynucleotides having sequences with a selected level of identity or homology to a complementary sequence of the particular polynucleotide probe. For example, in one aspect, the sequences having the selected level of identity or homology to the complementary sequence of the probe are identified by means of a BLAST or PowerBLAST algorithm. In various aspects, the plurality of polynucleotides consists of polynucleotides having sequences that are at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 99% identical to the complementary sequence of the particular polynucleotide probe. [0020] In still other embodiments, which are both more general and more preferred embodiments, the polynucleotide or oligonucleotide probes are ranked and/or selected according to a combination of two or more of the properties (a)-(h) listed above and, optionally, the sensitivity and/or specificity with which each probe hybridizes to a target polynucleotide. For example, in one embodiment the invention provides methods for selecting one or more different polynucleotide probes from a plurality of polynucleotide probes be a method comprising: (a) identifying those polynucleotide probes in the plurality of polynucleotide probes that have particular values (or a particular range of values) of one, two, three or more properties or parameters (e.g., selected among the properties and parameters listed hereinabove); and (b) selecting the polynucleotide probes identified in step (a). [0021] In another general embodiment, the methods of the invention comprise: (a) ranking the polynucleotide probes in a plurality of different polynucleotide probes according to each of two or more selected properties or parameters (e.g., selected from the properties and parameters recited hereinabove) so that a rank is obtained for each of the two or more selected parameters; and (b) obtaining a combined rank for each different polynucleotide probe, wherein the combined rank is determined from the sum of the ranks obtained for each of the two or more selected properties or parameters. One or more different polynucleotide probes can then be selected from the plurality of different polynucleotide probes according to the combined rank of the different polynucleotide probes. Continue reading about Iterative probe design and detailed expression profiling with flexible in-situ synthesis arrays... 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