Iterative probe design and detailed expression profiling with flexible in-situ synthesis arrays
First Claim
1. A method for selecting one or more different polynucleotide probes from a plurality of different polynucleotide probes of different nucleotide sequence according to the sensitivity and specificity with which each different polynucleotide probe hybridizes to a target polynucleotide, said method comprising:
- (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, wherein the specificity of a probe is represented by a quantity selected from the group consisting of (i) a ratio of target polynucleotide molecules to non-target polynucleotide molecules hybridized to said probe under a particular set of hybridization conditions and (ii) a cross-hybridization score based on calculated binding energies of said probe to non-target polynucleotide molecules, wherein said target polynucleotide molecules hybridize with said probe with perfect match, and wherein said non-target polynucleotide molecules hybridize with said probe with one or more mismatches; and
(c) selecting one or more different polynucleotide probes from the ranked polynucleotide probes.
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Abstract
Methods and compositions are provided that are useful for detecting and reporting a plurality of different target polynucleotide sequences in a sample, such as polynucleotides corresponding to a plurality of different genes expressed by a cell or cells. In particular, the invention provides methods for screening a plurality of candidate polynucleotide probes to evaluate both the sensitivity and the specificity with which each candidate probe hybridizes to a target polynucleoide sequence. Candidate polynucleotide probes can then be ranked according to both their sensitivity and specificity, and probes that have optimal sensitivity and specificity for a target polynucleotide sequence can be selected. In one embodiment, polynucleotide probes can be selected according to the methods described herein to prepare “screening chips” wherein a large number of target polynucleotide sequences are detected using a single microarray have a few (e.g., 1–5) probes for each target polynucleotide sequence. In a particularly preferred embodiment, the invention provides a screening chip that can detect genetic transcripts from the entire genome of an organism. In an alternative embodiment, polynucleotide probes can be selected according to the methods described herein to prepare “signature chips” to more accurately detect certain selected “signature genes” using several polynucleotide probes (e.g., 10–20) for each signature gene. The invention additionally provides microarrays containing polynucleotide probes for a large number of genes expressed by a cell or organism. Further, methods for detecting a plurality of polynucleotide molecules, including a large number of genes expressed by a cell or organism, are also provided.
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Citations
90 Claims
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1. A method for selecting one or more different polynucleotide probes from a plurality of different polynucleotide probes of different nucleotide sequence according to the sensitivity and specificity with which each different polynucleotide probe hybridizes to a target polynucleotide, said method comprising:
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(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, wherein the specificity of a probe is represented by a quantity selected from the group consisting of (i) a ratio of target polynucleotide molecules to non-target polynucleotide molecules hybridized to said probe under a particular set of hybridization conditions and (ii) a cross-hybridization score based on calculated binding energies of said probe to non-target polynucleotide molecules, wherein said target polynucleotide molecules hybridize with said probe with perfect match, and wherein said non-target polynucleotide molecules hybridize with said probe with one or more mismatches; and (c) selecting one or more different polynucleotide probes from the ranked polynucleotide probes. - View Dependent Claims (5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 42, 43, 44, 81)
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2. A method for selecting one or more different polynucleotide probes from a plurality of different polynucleotide probes of different nucleotide sequence according to the sensitivity and specificity with which each different polynucleotide probe hybridizes to a target polynucleotide, said method comprising:
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(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, wherein the specificity of a probe is represented by a quantity selected from the group consisting of (i) a ratio of target polynucleotide molecules to non-target polynucleotide molecules hybridized to said probe under a particular set of hybridization conditions and (ii) a cross-hybridization score based on calculated binding energies of said probe to non-target polynucleotide molecules, wherein said target polynucleotide molecules hybridize with said probe with perfect match, and wherein said non-target polynucleotide molecules hybridize with said probe with one or more mismatches; (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.
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3. A method for selecting one or more different polynucleotide probes from a plurality of different polynucleotide probes of different nucleotide sequence according to the sensitivity and specificity with which each different polynucleotide probe hybridizes to a target polynucleotide, said method comprising:
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(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, wherein the specificity of a probe is represented by a quantity selected from the group consisting of (i) a ratio of target polynucleotide molecules to non-target polynucleotide molecules hybridized to said probe under a particular set of hybridization conditions and (ii) a cross-hybridization score based on calculated binding energies of said probe to non-target polynucleotide molecules, wherein said target polynucleotide molecules hybridize with said probe with perfect match, and wherein said non-target polynucleotide molecules hybridize with said probe with one or more mismatches; (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. - View Dependent Claims (4)
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34. A method for selecting one or more different polynucleotide probes from a plurality of different polynucleotide probes of different nucleotide sequence according to the sensitivity and specificity with which each different polynucleotide probe hybridizes to a target polynucleotide, said method comprising:
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(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 polynucleotide sample correlates with the sensitivity and specificity with which each different polynucleotide probe hybridizes to the target polynucleotide, wherein the specificity of a probe is represented by a ratio of target polynucleotide molecules to non-target polynucleotide molecules hybridized to said probe under a particular set of hybridization conditions, wherein said target polynucleotide molecules hybridize with said probe with perfect match, and wherein said non-target polynucleotide molecules hybridize with said probe with one or more mismatches; and (b) selecting polynucleotide probes in the plurality of different polynucleotide probes that have the highest hybridization intensity. - View Dependent Claims (35, 36, 37, 38, 39, 40, 41)
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45. A system for selecting one or more different polynucleotide probes from a plurality of different polynucleotide probes of different nucleotide sequence according to the sensitivity and specificity with which each polynucleotide probe hybridizes to a target polynucleotide, said system comprising:
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(a) a memory; and (b) a processor element interconnected with the memory, wherein the memory encodes one or more programs causing the processor to perform a method comprising (i) identifying polynucleotide probes in the plurality of different polynucleotide probes that hybridize to the target polynucleotide with a sensitivity above a threshold sensitivity level; and (ii) ranking the identified polynucleotide probes according to the specificity with which each identified polynucleotide probe hybridizes to the target polynucleotide, wherein the specificity of a probe is represented by a quantity selected from the group consisting of (i) a ratio of target polynucleotide molecules to non-target polynucleotide molecules hybridized to said probe under a particular set of hybridization conditions and (ii) a cross-hybridization score based on calculated binding energies of said probe to non-target polynucleotide molecules, wherein said target polynucleotide molecules hybridize with said probe with perfect match, and wherein said non-target polynucleotide molecules hybridize with said probe with one or more mismatches. - View Dependent Claims (49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 67, 68, 82)
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46. A system for selecting one or more different polynucleotide probes from a plurality of different polynucleotide probes of different nucleotide sequence according to the sensitivity and specificity with which each polynucleotide probe hybridizes to a target polynucleotide, said system comprising:
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(a) a memory; and (b) a processor element interconnected with the memory, wherein the memory encodes one or more programs causing the processor to perform a method comprising (i) identifying polynucleotide probes in the plurality of different polynucleotide probes that hybridize to the target polynucleotide sequence with a specificity above a threshold specificity level, wherein the specificity of a probe is represented by a quantity selected from the group consisting of (i) a ratio of target polynucleotide molecules to non-target polynucleotide molecules hybridized to said probe under a particular set of hybridization conditions and (ii) a cross-hybridization score based on calculated binding energies of said probe to non-target polynucleotide molecules, wherein said target polynucleotide molecules hybridize with said probe with perfect match, and wherein said non-target polynucleotide molecules hybridize with said probe with one or more mismatches; and (ii) ranking the identified polynucleotide probes according to the sensitivity with which each identified polynucleotide probe hybridizes to the target polynucleotide.
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47. A system for selecting one or more different polynucleotide probes from a plurality of different polynucleotide probes of different nucleotide sequence according to the sensitivity and specificity with which each polynucleotide probe hybridizes to a target polynucleotide, said system comprising:
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(a) a memory; and (b) a processor element interconnected with the memory, wherein the memory encodes one or more programs causing the processor to perform a method comprising (i) 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; (ii) 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, wherein the specificity of a probe is represented by a quantity selected from the group consisting of (i) a ratio of target polynucleotide molecules to non-target polynucleotide molecules hybridized to said probe under a particular set of hybridization conditions and (ii) a cross-hybridization score based on calculated binding energies of said probe to non-target polynucleotide molecules, wherein said target polynucleotide molecules hybridize with said probe with perfect match, and wherein said non-target polynucleotide molecules hybridize with said probe with one or more mismatches; and (iii) 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. - View Dependent Claims (48)
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63. A computer program product for use in conjunction with a computer having a memory and a processor, the computer program product comprising a computer readable storage medium having a computer program mechanism encoded thereon, wherein said computer program mechanism may be loaded into the memory of a computer and cause a processor of the computer to execute the steps of:
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(a) identifying polynucleotide probes in a plurality of different polynucleotide probes of different nucleotide sequence that hybridize to a target polynucleotide with a sensitivity above a threshold level; and (b) ranking the identified polynucleotide probes according to the specificity with which each identified polynucleotide probe hybridizes to the target polynucleotide, wherein the specificity of a probe is represented by a quantity selected from the group consisting of (i) a ratio of target polynucleotide molecules to non-target polynucleotide molecules hybridized to said probe under a particular set of hybridization conditions and (ii) a cross-hybridization score based on calculated binding energies of said probe to non-target polynucleotide molecules, wherein said target polynucleotide molecules hybridize with said probe with perfect match, and wherein said non-target polynucleotide molecules hybridize with said probe with one or more mismatches. - View Dependent Claims (69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 83)
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64. A computer program product for use in conjunction with a computer having a memory and a processor, the computer program product comprising a computer readable storage medium having a computer program mechanism encoded thereon, wherein said computer program mechanism may be loaded into the memory of a computer and cause a processor of the computer to execute the steps of:
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(a) identifying polynucleotide probes in a plurality of different polynucleotide probes of different nucleotide sequence that hybridize to a target polynucleotide with a specificity above a threshold level, wherein the specificity of a probe is represented by a quantity selected from the group consisting of (i) a ratio of target polynucleotide molecules to non-target polynucleotide molecules hybridized to said probe under a particular set of hybridization conditions and (ii) a cross-hybridization score based on calculated binding energies of said probe to non-target polynucleotide molecules, wherein said target polynucleotide molecules hybridize with said probe with perfect match, and wherein said non-target polynucleotide molecules hybridize with said probe with one or more mismatches; and (b) ranking the identified polynucleotide probes according to the sensitivity with which each identified polynucleotide probe hybridizes to the target polynucleotide.
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65. A computer program product for use in conjunction with a computer having a memory and a processor, the computer program product comprising a computer readable storage medium having a computer program mechanism encoded thereon, wherein said computer program mechanism may be loaded into the memory of a computer and cause a processor of the computer to execute the steps of:
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(a) ranking a plurality of different polynucleotide probes of different nucleotide sequence according to the sensitivity with which each polynucleotide probe hybridizes to a 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, wherein the specificity of a probe is represented by a quantity selected from the group consisting of (i) a ratio of target polynucleotide molecules to non-target polynucleotide molecules hybridized to said probe under a particular set of hybridization conditions and (ii) a cross-hybridization score based on calculated binding energies of said probe to non-target polynucleotide molecules, wherein said target polynucleotide molecules hybridize with said probe with perfect match, and wherein said non-target polynucleotide molecules hybridize with said probe with one or more mismatches; and (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. - View Dependent Claims (66)
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84. A method for selecting one or more different polynucleotide probes from a plurality of different polynucleotide probes of different nucleotide sequence according to the sensitivity and specificity with which each different polynucleotide probe hybridizes to a target polynucleotide, said method comprising:
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(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, wherein the specificity of a probe is represented by a ratio of target polynucleotide molecules to non-target polynucleotide molecules hybridized to said probe under a particular set of hybridization conditions, wherein said target polynucleotide molecules hybridize with said probe with perfect match, and wherein said non-target polynucleotide molecules hybridize with said probe with one or more mismatches, and wherein the ratio is calculated from quantities provided by a method that comprises; (i) determining the level of hybridization of the target polynucleotide to the particular polynucleotide probe; and (ii) determining the level of cross hybridization of non-target polynucleotides to the particular polynucleotide probe; and (c) selecting one or more different polynucleotide probes from the ranked polynucleotide probes. - View Dependent Claims (87, 88, 89, 90)
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85. A method for selecting one or more different polynucleotide probes from a plurality of different polynucleotide probes of different nucleotide sequence according to the sensitivity and specificity with which each different polynucleotide probe hybridizes to a target polynucleotide, said method comprising:
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(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, wherein the specificity of a probe is represented by a ratio of target polynucleotide molecules to non-target polynucleotide molecules hybridized to said probe under a particular set of hybridization conditions, wherein said target polynucleotide molecules hybridize with said probe with perfect match, and wherein said non-target polynucleotide molecules hybridize with said probe with one or more mismatches, and wherein the ratio is calculated from quantities provided by a method that comprises; (i) determining the level of hybridization of the target polynucleotide to the particular polynucleotide probe; and (ii) determining the level of cross hybridization of non-target polynucleotides to the particular polynucleotide probe; (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.
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86. A method for selecting one or more different polynucleotide probes from a plurality of different polynucleotide probes of different nucleotide sequence according to the sensitivity and specificity with which each different polynucleotide probe hybridizes to a target polynucleotide, said method comprising:
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(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, wherein the specificity of a probe is represented by a ratio of target polynucleotide molecules to non-target polynucleotide molecules hybridized to said probe under a particular set of hybridization conditions, wherein said target polynucleotide molecules hybridize with said probe with perfect match, and wherein said non-target polynucleotide molecules hybridize with said probe with one or more mismatches, and wherein the ratio is calculated from quantities provided by a method that comprises; (i) determining the level of hybridization of the target polynucleotide to the particular polynucleotide probe; and (ii) determining the level of cross hybridization of non-target polynucleotides to the particular 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.
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Specification