Iterative probe design and detailed expression profiling with flexible in-situ synthesis arrays

• US 7,013,221 B1
• Filed: 04/28/2000
• Issued: 03/14/2006
• Est. Priority Date: 07/16/1999
• Status: Expired due to Term

• Current Assignee: Merck & Co., Inc.
• Original Assignee: Rosetta Inpharmatics LLC (Merck & Co., Inc.)
• Inventors: Stoughton, Roland, Linsley, Peter S., Friend, Stephen H., Burchard, Julja
• Primary Examiner(s): Allen, Marianne P.
• Application Number:
  US09/561,487
• Time in Patent Office: 2,146 Days
• Field of Search: 702/19, 702/20, 435/6
• US Class Current: 702/20
• CPC Class Codes: G16B 25/00 ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
• CPC Class Codes: G16B 25/10 Gene or protein expression profiling; Expression-ratio estimation or normalisation
• CPC Class Codes: G16B 25/20 Polymerase chain reaction [PCR]; Primer or probe design; Probe optimisation

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.

Claims

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.

5. The method of any one of claims 1–

3 wherein the sensitivity with which a particular polynucleotide probe hybridizes to the target polynucleotide is represented by the binding energy with which the target polynucleotide hybridizes to the particular polynucleotide probe.

6. The method of claim 5 wherein the binding energy with which the target polynucleotide hybridizes to the particular polynucleotide probe is determined according to a nearest neighbor model.

7. The method of any one of claims 1–

3 wherein the sensitivity with which a particular polynucleotide probe hybridizes to the target polynucleotide is represented by the level of hybridization of a target polynucleotide sequence to the particular polynucleotide probe.

8. The method of claim 7 wherein the level of hybridization of the target polynucleotide to the particular polynucleotide probe is calculated from the binding energy with which the target polynucleotide hybridizes to the particular polynucleotide probe.

9. The method of claim 8 wherein the binding energy with which the target polynucleotide hybridizes to the particular polynucleotide probe sequence is determined according to a nearest neighbor model.

10. The method of any one of claims 1–

3 wherein the sensitivity of a particular polynucleotide probe is represented by the level of hybridization of a target polynucleotide to the particular polynucleotide probe, and wherein said level of hybridization is provided by a method comprising measuring the level of hybridization of the target polynucleotide to the particular polynucleotide probe.

11. The method of any one of claims 1–

3, wherein the specificity of a particular probe is represented by said cross-hybridization score, and wherein the the cross-hybridization score is provided by a method which comprises selecting, from a plurality of binding energies, a binding energy that is the most negative binding energy in said plurality of binding energies,

wherein each binding energy in said plurality of binding energies is a binding energy for hybridization of the particular polynucleotide probe to a different polynucleotide, each different polynucleotide also differing from the target polynucleotide, and

wherein the selected binding energy is said cross-hybridization score of the particular polynucleotide probe.

12. The method of claim 11, wherein the binding energy for hybridization of the particular polynucleotide probe to each of the different polynucleotides is determined according to a nearest neighbor model.

13. The method of claim 11, wherein the different polynucleotides comprises polynucleotides expressed by a cell or organism of interest.

14. The method of claim 11, wherein the different polynucleotides consist of polynucleotides comprising sequences with a selected level of identity or homology to a complementary sequence of said particular polynucleotide probe.

15. The method of claim 14, wherein 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.

16. The method of claim 14, wherein the different polynucleotides consist of polynucleotides comprising sequences that are at least 50% identical to the complementary sequence of said particular polynucleotide probe.

17. The method of claim 14, wherein the different polynucleotides consist of polynucleotides comprising sequences that are at least 60% identical to the complementary sequence of said particular polynucleotide probe.

18. The method of claim 14, wherein the different polynucleotides consist of polynucleotides comprising sequences that are at least 70% identical to the complementary sequence of said particular polynucleotide probe.

19. The method of claim 14, wherein the different polynucleotides consist of polynucleotides comprising sequences that are at least 80% identical to the complementary sequence of said particular polynucleotide probe.

20. The method of claim 14, wherein the different polynucleotides consist of polynucleotides comprising sequences that are at least 90% identical to the complementary sequence of said particular polynucleotide probe.

21. The method of claim 14, wherein the different polynucleotides consist of polynucleotides comprising sequences that are at least 95% identical to the complementary sequence of said particular polynucleotide probe.

22. The method of claim 14, wherein the different polynucleotides consist of polynucleotides comprising sequences that are at least 99% identical to the complementary sequence of said particular polynucleotide probe.

23. The method of any one of claims 1–

3, said method further comprising a step of rejecting polynucleotide probes comprising one or more sequences corresponding to a repetitive element or a simple repeat.

24. The method of any one of claims 1–

3, said method further comprising a step of rejecting polynucleotide probes comprising one or more sequences corresponding to a polyX repeat.

25. The method of any one of claims 1–

3, wherein each of the plurality of different polynucleotide probes is complementary to a sequence in a region of the target polynucleotide.

26. The method of claim 25, wherein said region of the target polynucleotide is within a selected distance from the 3′

-end of the target polynucleotide.

27. The method of claim 25, wherein said region of the target polynucleotide is within a selected distance from the 5′

-end of the target polynucleotide.

28. The method of any one of claims 1–

3, further comprising a step of rejecting polynucleotide probes having a fraction of one or more particular nucleotide bases that is above a particular threshold.

29. The method of any one of claims 1–

3, further comprising a step of rejecting polynucleotide probes having a fraction of one or more particular nucleotide bases that is below a particular threshold.

30. The method of claim 28, wherein said step of rejecting comprises rejecting polynucleotide probes having a fraction of guanine and cytosine bases above a particular threshold.

31. The method of any one of claims 1–

3, further comprising a step of rejecting polynucleotide probes for which a particular mathematical combination of the fraction of two or more particular nucleotide bases present in the polynucleotide probe is above a particular threshold.

32. The method of claim 31 wherein said step of rejecting comprises rejecting polynucleotide probes for which the difference in the fraction of nucleotide bases present in the polynucleotide probe that are cytosine from the fraction of nucleotide bases present in the polynucleotide probe that are adenine is above a particular threshold.

33. The method of any one of claims 1–

3, further comprising a step of rejecting polynucleotide probes for which a particular mathematical combination of the fraction of two or more particular nucleotide bases present in the polynucleotide probe is below a particular threshold.

42. The method of any one of claims 1–

3 or 34 wherein the plurality of different polynucleotide probes comprises polynucleotide sequences between 15 and 500 bases in length.

43. The method of claim 42 wherein the polynucleotide sequences are between 20 and 100 bases in length.

44. The method of claim 43 wherein the polynucleotide sequences are between 40 and 60 bases in length.

81. The method of any one of claims 1–

3, further comprising a step of rejecting polynucleotide probes having 100% sequence identity to a sequence that is not part of the sequence of said target polynucleotide.

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:

(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.

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:

(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.

4. The method of claim 3 wherein the sum of the sensitivity rank and the specificity rank for each different polynucleotide probe is a weighted sum of the sensitivity rank and the specificity rank for each different polynucleotide probe.

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:

(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.

35. The method of claim 34 wherein the hybridization temperature is within 5°

C. of the mean melting temperature of the plurality of different polynucleotide probes from the target polynucleotide.

36. The method of claim 35 wherein the hybridization temperature is within 2°

C. of the mean melting temperature of the plurality of different polynucleotide probes from the target polynucleotide.

37. The method of claim 34 wherein the steps of hybridizing and selecting are iteratively repeated.

38. The method of claim 37 wherein the number of different polynucleotide probes selected is no more than 20.

39. The method of claim 38 wherein the number of different polynucleotide probes selected is no more than 10.

40. The method of claim 39 wherein the number of different polynucleotide probes selected is no more than 5.

41. The method of claim 40 wherein one polynucleotide probe is selected from the plurality of different polynucleotide probes.

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:

(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.

49. The system of any one of claims 45–

47 wherein the sensitivity with which a particular polynucleotide probe hybridizes to the target polynucleotide is represented by the binding energy with which said particular polynucleotide probe hybridizes to the target polynucleotide.

50. The system of claim 49 wherein the programs cause the binding energy with which the target polynucleotide hybridizes to the particular polynucleotide probe to be calculated according to a nearest neighbor model.

51. The system of any one of claims 45–

47, wherein the specificity of a particular probe is represented by said cross-hybridization score, and wherein said programs further cause the processor to determine the cross-hybridization score by a method which comprises selecting, from a plurality of binding energies, a binding energy that is the most negative binding energy in said plurality of binding energies,

wherein each binding energy in said plurality of binding energies is a binding energy for hybridization of the particular polynucleotide probe to a different polynucleotide, each different polynucleotide also differing from the target polynucleotide, and

wherein the selected binding energy is said cross-hybridization score of the particular polynucleotide probe.

52. The system of claim 51 wherein said programs further cause the processor to perform steps of calculating the binding energies in said plurality of binding energies according to a nearest neighbor model.

53. The system of claim 51 wherein the different polynucleotides consist of polynucleotides comprising sequences with a selected level of identity or homology to a complementary sequence of said particular polynucleotide probe.

54. The system of claim 53 wherein said programs cause the processor to identify sequences having the selected level of identity or homology to a complementary sequence of said particular polynucleotide probe by means of a BLAST or PowerBLAST algorithm.

55. The system of any one of claims 45–

47 wherein said programs further cause the processor to perform a step of rejecting polynucleotide probes comprising one or more sequences corresponding to a repetitive element or a simple repeat.

56. The system of any one of claims 45–

47 wherein said programs further cause the processor to perform a step of rejecting polynucleotide probes comprising one or more sequences corresponding to a polyX repeat.

57. The system of any one of claims 45–

47 wherein said programs further cause the processor to perform a step of selecting polynucleotide probes comprising a sequence that is complementary to a sequence within a particular distance from the 3′

-end of the target polynucleotide.

58. The system of any one of claims 45–

47 wherein said programs further cause the processor to perform a step of selecting polynucleotide probes comprising a sequence that is complementary to a sequence within a particular distance from the 5′

-end of the target polynucleotide.

59. The system of any one of claims 45–

47 wherein said programs further cause the processor to perform a step of rejecting polynucleotides probes having a fraction of one or more particular nucleotide bases that is above a particular threshold.

60. The system of any one of claims 45–

47 wherein said programs further cause the processor to perform a step of rejecting polynucleotide probes having a fraction of one or more particular nucleotide bases that is below a particular threshold.

61. The system of any one of claims 45–

47 wherein said programs further cause the processor to perform a step of rejecting polynucleotide probes for which a particular mathematical combination of the fraction of two or more particular nucleotide bases present in the polynucleotide probes is above a particular threshold.

62. The system of any one of claims 45–

47 wherein said programs further cause the processor to perform a step of rejecting polynucleotide probes for which a particular mathematical combination of the fraction of two or more particular nucleotide bases present in the polynucleotide probes is below a particular threshold.

67. The computer program product of any one of claims 62–

65 wherein the sensitivity with which a particular polynucleotide probe hybridizes to the target polynucleotide is represented by the binding energy with which said particular polynucleotide probe hybridizes to the target polynucleotide.

68. The computer program product of claim 67 wherein said computer program mechanism may be loaded into the memory and further cause the binding energy with which the target polynucleotide hybridizes to the particular polynucleotide probe to be calculated according to a nearest neighbor model.

82. The system of any one of claims 45–

47, wherein said programs further cause the processor to perform a step of rejecting polynucleotide probes having 100% sequence identity to a sequence that is not part of the sequence of said target polynucleotide.

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:

(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.

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:

(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.

48. The system of claim 47 wherein the sum of the sensitivity rank and the specificity rank for each different polynucleotide probe is a weighted sum of the sensitivity rank and the specificity rank for each different polynucleotide probe.

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:

(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.

69. The computer program product of any one of claim 63–

65 wherein the specificity of a particular probe is represented by said cross-hybridization score, and wherein said computer program mechanism can be loaded into the memory and further cause the processor to determine the cross-hybridization score by a method which comprises selecting, from a plurality of binding energies, a binding energy that is the most negative binding energy in said plurality of binding energies,

wherein each binding energy in said plurality of binding energies is a binding energy for hybridization of the particular polynucleotide probe to a different polynucleotide, each different polynucleotide also differing from the target polynucleotide, and

wherein the selected binding energy is said cross-hybridization score of the particular polynucleotide probe.

70. The computer program product of claim 69 wherein said computer program mechanism can be loaded into the memory and further cause the processor to perform steps of calculating the binding energies in said plurality of binding energies according to a nearest neighbor model.

71. The computer program product of claim 69 wherein the different polynucleotides consist of polynucleotides comprising sequences with a selected level of identity or homology to a complementary sequence of said particular polynucleotide probe.

72. The computer program product of claim 71 wherein said computer program mechanism can be loaded into the memory and further cause the processor to identify sequences having the selected level of identity or homology to a complementary sequence of said particular polynucleotide probe by means of a BLAST or PowerBLAST algorithm.

73. The computer program product of any one of claims 63–

65 wherein said computer program mechanism can be loaded into the memory and further cause the processor to perform a step of rejecting polynucleotide probes comprising one or more sequences corresponding to a repetitive element or a simple repeat.

74. The computer program product of any one of claims 63–

65 wherein said computer program mechanism can be loaded into the memory and further cause the processor to perform a step of rejecting polynucleotide probes comprising one or more sequences corresponding to a polyX repeat.

75. The computer program product of any one of claims 63–

65 wherein said computer program mechanism can be loaded into the memory and further cause the processor to perform a step of selecting polynucleotide probes comprising a sequence that is complementary to a sequence within a particular distance from the 3′

-end of the target polynucleotide.

76. The computer program product of any one of claims 63–

65 wherein said computer program mechanism can be loaded into the memory and further cause the processor to perform a step of selecting polynucleotide probes comprising a sequence that is complementary to a sequence within a particular distance from the 5′

-end of the target polynucleotide.

77. The computer program product of any one of claims 63–

65 wherein said computer program mechanism can be loaded into the memory and further cause the processor to perform a step of rejecting polynucleotides probes having a fraction of one or more particular nucleotide bases that is above a particular threshold.

78. The computer program product of any one of claims 63–

65 wherein said computer program mechanism can be loaded into the memory and further cause the processor to perform a step of rejecting polynucleotide probes having a fraction of one or more particular nucleotide bases that is below a particular threshold.

79. The computer program product of any one of claims 63–

65 wherein said computer program mechanism can be loaded into the memory and further cause the processor to perform a step of rejecting polynucleotide probes for which a particular mathematical combination of the fraction of two or more particular nucleotide bases present in the polynucleotide probes is above a particular threshold.

80. The computer program product of any one of claims 63–

65 wherein said computer program mechanism can be loaded into the memory and further cause the processor to perform a step of rejecting polynucleotide probes for which a particular mathematical combination of the fraction of two or more particular nucleotide bases present in the polynucleotide probes is below a particular threshold.

83. The computer program product of any one of claims 63–

65, wherein said computer program mechanism can be loaded into the memory and further cause the processor to perform a step of rejecting polynucleotide probes having 100% sequence identity to a sequence that is not part of the sequence of said target polynucleotide.

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:

(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.

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:

(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.

66. The computer program product of claim 65 wherein the sum of the sensitivity rank and the specificity rank for each different polynucleotide probe is a weighted sum of the sensitivity rank and the specificity rank for each different polynucleotide probe.

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:

(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.

87. The method of claim 84, 85 or 86, wherein the level of hybridization of the target polynucleotide to the particular polynucleotide probe is calculated from the binding energy with which the target polynucleotide hybridizes to the particular polynucleotide probe.

88. The method of claim 84, 85 or 86 wherein the binding energy with which the target polynucleotide hybridizes to the particular polynucleotide probe is provided according to a nearest neighbor model.

89. The method of claim 84, 85 or 86, wherein the level of cross hybridization of non-target polynucleotides to the particular polynucleotide probe is calculated from the binding energies with which non-target polynucleotides cross-hybridize to the particular polynucleotide probe.

90. The method of claim 89 wherein the binding energies with which non-target polynucleotides cross-hybridize to the particular polynucleotide probe are determined according to a nearest neighbor model.

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:

(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.

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:

(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.