METHODS AND COMPUTER SYSTEMS FOR IDENTIFYING TARGET-SPECIFIC SEQUENCES FOR USE IN NANOREPORTERS

• US 20100112710A1
• Filed: 04/10/2008
• Published: 05/06/2010
• Est. Priority Date: 04/10/2007
• Status: Active Grant

• Current Assignee: Nanostring Technologies, Inc.
• Original Assignee: Nanostring Technologies, Inc.
• Inventors: Dahl, Tim, Dahl, Craig E., Geiss, Gary K., Davidson, Eric H.
• Granted Patent: US 8,415,102 B2
• Time in Patent Office: Days
• US Class Current: 436/94
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: G16B 30/00 ICT specially adapted for sequence analysis involving nucleotides or amino acids
• CPC Class Codes: G16B 30/10 Sequence alignment; Homology search
• CPC Class Codes: G16B 30/20 Sequence assembly
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

The present invention relates to compositions and methods for detection and quantification of individual target molecules in biomolecular samples. In particular, the invention relates to coded, labeled probes that are capable of binding to and identifying target molecules based on the probes'"'"' label codes. Methods, computers, and computer program products for identifying target-specific sequences for inclusion in the probes are also provided, as are methods of making and using such probes. The probes can be used in diagnostic, prognostic, quality control and screening applications.

Claims

1. A method for identifying a pair of adjacent target-specific sequences for use in a probe pair hybridizable to a target mRNA, comprising the steps of:

(a) generating a first pool of candidate nucleotide sequences of a first predetermined length or lengths that are reverse complements of a target mRNA sequence, wherein each candidate nucleotide sequence can be divided into two adjacent nucleotide sequences of equal length consisting of a 5′

candidate sequence and a 3′

candidate sequence;

(b) deleting from said first pool one or more candidate nucleotide sequences that meet at least two of the following criteria;

(i) contain inverted repeats of greater than a predetermined length of consecutive nucleotides;

(ii) contain direct repeats of greater than a predetermined length of consecutive nucleotides;

(iii) whose 5′

candidate sequence and/or 3′

candidate sequence have a GC content outside a predetermined range;

(iv) whose 5′

candidate sequence and/or 3′

candidate sequence contain contiguous stretches of C residues of greater than a predetermined length; and

(v) whose 5′

candidate sequence and/or 3′

candidate sequence have melting temperatures that are outside a first predetermined melting temperature range;

thereby generating a second pool of candidate nucleotide sequences;

(c) deleting from said second pool one or more candidate nucleotide sequences whose 5′

candidate sequence and/or 3′

candidate sequence has a cross-hybridization potential to non-specific sequences that is higher than a predetermined threshold, thereby generating a third pool of candidate nucleotide sequences;

(d) deleting from said third pool one or more candidate nucleotide sequences whose 5′

candidate sequence and/or 3′

candidate sequence has a melting temperature outside a second predetermined temperature range, wherein the second predetermined melting temperature range is within the first predetermined melting temperature range;

(e) determining the melting temperature for a modified 5′

candidate sequence or a modified 3′

candidate sequence, wherein the modified 5′

candidate sequence or a modified 3′

candidate sequence is a modified form of a 5′

candidate sequence or a 3′

candidate sequence, respectively, of a candidate nucleotide sequence deleted in step (d) because its 5′

candidate sequence and/or 3′

candidate sequence has a melting temperature above the second predetermined range, wherein the modified 5′

candidate sequence has been modified by trimming at least one nucleotide from the 5′

end of the corresponding 5′

candidate sequence, and wherein the modified 3′

candidate sequence has been modified by trimming at least one nucleotide from the 3′

end of the corresponding 3′

candidate sequence;

(f) in the event that;

(A) the modified 5′

or modified 3′

candidate sequence, and

(B) a 3′

or 5′

, respectively, candidate sequence or the modified form thereof;

each have a melting temperature within the second predetermined melting temperature range and both are derived from the same candidate nucleotide sequence;

adding to the third pool a modified candidate nucleotide sequence composed of (A) and (B), thereby generating a fourth pool of candidate nucleotide sequences;

(g) in the event that the length of the modified 5′

or modified 3′

candidate sequence is greater than a second predetermined length, repeating step (e) one or more times wherein the modified 5′

candidate sequence or modified 3′

candidate sequence, respectively, has been trimmed by a greater number of nucleotides than in step (e) each time, until the length of the modified 5′

or modified 3′

candidate sequence is the earlier of (i) equal to, or (ii) lower than, the second predetermined length;

(h) for each modified 5′

or modified 3′

candidate sequence of step (g) wherein;

(C) said modified 5′

or modified 3′

candidate sequence, and

(D) a 3′

or 5′

, respectively, candidate sequence or the modified form thereof;

each have a melting temperature within the second predetermined melting temperature range and both are derived from the same candidate nucleotide sequence;

adding to the third pool a modified candidate sequence composed of (C) and (D), thereby generating a fifth pool of candidate nucleotide sequences; and

(i) optionally repeating steps (e)-(h) for one or more different candidate nucleotide sequences deleted in step (d),

thereby generating a sixth pool of candidate nucleotide sequences,

whereby the fourth, fifth and sixth pools consist of candidate nucleotide sequences composed of pairs of adjacent target-specific sequences for use in a probe pair hybridizable to the target mRNA.

2. The method of claim 1, which further comprises the step of outputting to a user interface device, a computer readable storage medium, or a local or remote computer system, or displaying, one or a plurality of candidate nucleotide sequences and/or modified candidate nucleotide sequences in the fourth, fifth and/or sixth pools and/or the 5′

candidate sequences or modified 5′

candidate sequences and/or 3′

candidate sequences or modified 3′

candidate sequences contained therein.

3. The method of claim 2, wherein the candidate nucleotide sequences and/or modified candidate nucleotide sequences are outputted as pairs of adjacent target-specific nucleotide sequences derived from said candidate nucleotide sequences and/or modified candidate nucleotide sequences, respectively.

4. The method of any one of claims 1 to 3, wherein in step (c) a 5′

candidate sequence or 3′

candidate sequence is deemed to have a cross-hybridization potential to non-specific sequences that is higher than said predetermined threshold if said 5′

candidate sequence or 3′

candidate sequence has (i) a sequence percentage identity with a first sequence (hereinafter “

first non-target sequence”

) or its complement that is equal to or greater than a first predetermined cutoff, said first non-target sequence being other than the complement of the target mRNA and, optionally, other than the complements of one or more alternatively spliced mRNAs corresponding to the same gene as the target mRNA, and said first non-target sequence being present in a database comprising cellular mRNA sequences or cDNA sequences derived therefrom; and

(ii) a contiguous block of sequence identity with a second sequence (hereinafter “

second non-target sequence”

) or its complement that is equal to or greater than a second predetermined cutoff, said second non-target sequence being other than the complement of the target mRNA and, optionally, other than the complements of one or more alternatively spliced mRNAs corresponding to the same gene as the target mRNA, and said second non-target sequence being present in the database.

5. The method of any one of claims 1 to 4, wherein a plurality of candidate nucleotide sequences and/or modified candidate nucleotide sequences in the fourth, fifth and/or sixth pools and/or the 5′

candidate sequences or modified 5′

candidate sequences and 3′

candidate sequences or modified 3′

candidate sequences contained therein are outputted or displayed in a ranked order based on a weighted score of the cross-hybridization potentials and the melting temperatures of said 5′

candidate sequences or modified 5′

candidate sequences and 3′

candidate sequences or modified 3′

candidate sequences.

6. The method of claim 5, wherein the weighted score is calculated according to the formula:

(Tm score*WFa)+(MCB score*WFb)+(PID score*WFc)

where;

Tm score is a melting temperature score calculated according to the formula;

(differential score+general score)/3

where the differential score is calculated according to the following formula;

1−

|(TmA−

TmB)|/(TmHco−

TmLco)

where the general score is calculated according to the following formula;

(((TmI−

|(TmA−

TmI)|)/TmI)+(((TmI−

|(TmB−

TmI)|)/TmI|)))

where TmA is the melting temperature of the 5′

candidate sequence or modified 5′

candidate sequence in a pair of adjacent target-specific sequences, TmB is the melting temperature of the 3′

candidate sequence or modified 3′

candidate sequence in said pair of adjacent target-specific sequences, TmHco is the upper limit of the second predetermined temperature range;

TmLco is the lower limit of the second predetermined temperature range; and

TmI is a predetermined ideal melting temperature;

where;

MCB score is a maximum contiguous block score calculated according to the formula;

1−

(MCB/MCBco);

where MCB is the greater of;

(i) the maximum contiguous block of identity between;

(A) a first target-specific nucleotide sequence in said pair of adjacent target-specific sequences; and

(B) a sequence in the database other than the complement of the target mRNA and, optionally, other than the complements of one or more alternatively spliced mRNAs corresponding to the same gene as the target mRNA;

and

(ii) the maximum contiguous block of identity between;

(A) a second target-specific nucleotide sequence in said pair of adjacent target-specific sequences; and

(B) a sequence in the database other than the complement of the target mRNA and, optionally, other than the complements of one or more alternatively spliced mRNAs corresponding to the same gene as the target mRNA,

and wherein MCBco is the first predetermined cutoff;

where;

PID score is a percent identity score calculated according to the formula;

1−

(PID/PIDco));

where PID is the greater of;

(i) the greatest percentage sequence identity between;

(A) a first target-specific nucleotide sequence in said pair of adjacent target-specific sequences; and

(B) a sequence in the database other than the complement of the target mRNA and, optionally, other than the complements of one or more alternatively spliced mRNAs corresponding to the same gene as the target mRNA;

and

(ii) the greatest percentage sequence identity between;

(A) a second target-specific nucleotide sequence in said pair of adjacent target-specific sequences; and

(B) a sequence in the database other than the complement of the target mRNA and, optionally, other than the complements of one or more alternatively spliced mRNAs corresponding to the same gene as the target mRNA,

and wherein PIDco is the second predetermined cutoff,

and where WFa, WFb, and WFc are each independently a weighting factor, each of which is a real number.

12. A method for identifying a plurality of pairs of adjacent target-specific sequences for use in a respective plurality of probe pairs, each probe pair being hybridizable to a different target mRNA, comprising, for each target mRNA:

identifying a pair of adjacent target-specific sequences according to the method of any one of claims 1 to 11.

18. A method for identifying a plurality of target-specific sequences for use in a respective plurality of probes, each probe being hybridizable to a different target mRNA, comprising, for each target mRNA:

identifying a target-specific sequence according to the method of any one of claims 1 to 17.

19. The method of any one of claims 1 to 18 wherein if the fourth, fifth and/or sixth pools contains no candidate nucleotide sequences, the method further comprises repeating steps (b) to (i), wherein step (b) is performed under more relaxed criteria.

20. The method of any one of claims 1 to 12, wherein in each step (g) of repeating the modified 5′

candidate sequence or modified 3′

candidate sequence is trimmed by one nucleotide.

22. The method of any one of claims 1 to 6 and 13 to 18, wherein step (b) comprises deleting from said first pool one or more candidate nucleotide sequences that meet at least 3 of said following criteria.

23. The method of claim 22, wherein step (b) comprises deleting from said first pool one or more candidate nucleotide sequences that meet 4 of said following criteria.

24. The method of claim 22, wherein step (b) comprises deleting from said first pool one or more candidate nucleotide sequences that meet all 5 of said following criteria.

25. The method of claim any one of claims 1 to 6, wherein the first predetermined length is selected from the range of 70 to 120 nucleotides.

26. The method of claim 25, wherein the second predetermined length is selected from the range of 30 to 45 nucleotides.

29. The method of any one of claims 1 to 6 and 13 to 18, wherein the predetermined length of the inverted repeats of step (b)(i) is selected from the range of 5 to 7 consecutive nucleotides.

30. The method of any one of claims 1 to 6 and 13 to 18, wherein the predetermined length of the direct repeats of step (b)(ii) is selected from the range of 7 to 9 consecutive nucleotides.

31. The method of any one of claims 1 to 6 and 13 to 18, wherein the predetermined range of GC content of step (b)(iii) is from 35-45% at the lower limit to 65-80% at the upper limit.

32. The method of claim 31, wherein the predetermined range of GC content of step (b)(iii) is 40-70%.

33. The method of any one of claims 1 to 6 and 13 to 18, wherein the predetermined length in step (b)(iv) is 3.

34. The method of any one of claims 1 to 6 and 13 to 18, wherein the highest and lowest temperatures of the first predetermined melting temperature range differ by 20°

C. to 25°

C.

35. The method of claim 34, wherein the first predetermined melting temperature range is from 60°

C. to 90°

C.

36. The method of claim 34, wherein the first predetermined melting temperature range is from 65°

C. to 85°

C.

41. The method of any one of claims 1 to 6 and 13 to 18, wherein the highest and lowest temperatures of the second predetermined melting temperature range differ by 4°

C. to 8°

C.

42. The method of claim 41, wherein the second predetermined melting temperature range is from 78°

C. to 83°

C.

44. The method of any one of claims 1 to 43, which further comprises the step of deleting from the fourth, fifth and/or sixth pools candidate nucleotide sequences that have a cross-hybridization potential to sequences present in other components of the probe or in a preparation step for the probe.

45. The method of any one of claims 1 to 43, wherein the target mRNA is an alternatively spliced mRNA.

46. The method of claim 45, which further comprises the step of determining whether one or more candidate nucleotide sequences are unique to one splice form or common to more than one splice form of the target mRNA.

47. The method of claim 45, wherein the first pool of candidate nucleotide sequences contains only candidate nucleotide sequences unique to one splice form.

48. The method of claim 45, wherein the first pool of candidate target-specific sequences contains only candidate nucleotide sequences common to multiple splice forms.

49. The method of any one of claims 1 to 48, which is a computer-implemented method.

50. A computer system for identifying a pair of adjacent target-specific sequences for use in a probe pair hybridizable to a target mRNA, comprising:

a processor and a memory coupled with the processor, the memory storing a plurality of machine instructions that cause the processor to perform the method of any one of claims 1 to 10.

51. A computer system for identifying a plurality of pairs of adjacent target-specific sequences for use in a respective plurality of probe pairs, each probe pair being hybridizable to a different target mRNA, comprising:

a processor and a memory coupled with the processor, the memory storing a plurality of machine instructions that cause the processor to perform the method of claim 12.

53. A computer system for identifying a plurality of target-specific sequences for use in a respective plurality of probes, each probe being hybridizable to a different target mRNA, comprising:

a processor and a memory coupled with the processor, the memory storing a plurality of machine instructions that cause the processor to perform the method of claim 18.

54. A computer system comprising:

a processor and a memory coupled with the processor, the memory storing a plurality of machine instructions that cause the processor to perform the method of any one of claims 1 to 49.

55. A computer program product for use in conjunction with a computer system, the computer program product comprising a computer readable storage medium and a computer program mechanism embedded therein, the computer program mechanism comprising instructions for performing the method of any one of claims 1 to 49.

7. A method for identifying a pair of adjacent target-specific sequences for use in a probe pair hybridizable to a target mRNA, comprising the steps of:

(a) generating a first pool of candidate nucleotide sequences of 100 nucleotides that are reverse complements of a target mRNA sequence, wherein each candidate nucleotide sequence can be divided into two adjacent nucleotide sequences of 50 nucleotides each, said adjacent nucleotide sequences consisting of a 5′

candidate sequence and a 3′

candidate sequence;

(b) deleting from said first pool one or more candidate nucleotide sequences that meet the following criteria;

(i) contain inverted repeats that are 6 consecutive nucleotides in length or greater;

(ii) contain direct repeats that are 9 consecutive nucleotides in length or greater;

(iii) whose 5′

candidate sequence and/or 3′

candidate sequence have a GC content outside 40-70%;

(iv) whose 5′

candidate sequence and/or 3′

candidate sequence contain contiguous stretches of 3 C residues or greater; and

(v) whose 5′

candidate sequence and/or 3′

candidate sequence have melting temperatures that are outside a range of (A) 60-90°

C. or (B) 65-85°

C.;

thereby generating a second pool of candidate nucleotide sequences;

(c) deleting from said second pool one or more candidate nucleotide sequences whose 5′

candidate sequence and/or 3′

candidate sequence has (i) a sequence percentage identity of 85% or greater with a first sequence (hereinafter “

first non-target sequence”

) or its complement, said first non-target sequence being other than the complement of the target mRNA and, optionally, other than the complements of one or more alternatively spliced mRNAs corresponding to the same gene as the target mRNA, and said first non-target sequence being present in a database comprising cellular mRNA sequences or cDNA sequences derived therefrom; and

(ii) a contiguous block of sequence identity of 15 nucleotides or greater with a second sequence (hereinafter “

second non-target sequence”

) or its complement, said second non-target sequence being other than the complement of the target mRNA and, optionally, other than the complements of one or more alternatively spliced mRNAs corresponding to the same gene as the target mRNA, and said second non-target sequence being present in the database;

thereby generating a third pool of candidate nucleotide sequences;

(d) deleting from said third pool one or more candidate nucleotide sequences whose 5′

candidate sequence and/or 3′

candidate sequence has a melting temperature outside the range of 78-83°

C.;

(e) determining the melting temperature for a modified 5′

candidate sequence or a modified 3′

candidate sequence, wherein the modified 5′

candidate sequence or a modified 3′

candidate sequence is a modified form of a 5′

candidate sequence or a 3′

candidate sequence, respectively, of a candidate nucleotide sequence deleted in step (d) because its 5′

candidate sequence and/or 3′

candidate sequence has a melting temperature above 83°

C., wherein the modified 5′

candidate sequence has been modified by trimming at least one nucleotide from the 5′

end of the corresponding 5′

candidate sequence, and wherein the modified 3′

candidate sequence has been modified by trimming at least one nucleotide from the 3′

end of the corresponding 3′

candidate sequence;

(f) in the event that;

(A) the modified 5′

or modified 3′

candidate sequence, and

(B) a 3′

or 5′

, respectively, candidate sequence or the modified form thereof,

each have a melting temperature within the range of 78-83°

C. and both are derived from the same candidate nucleotide sequence,

adding to the third pool a modified candidate nucleotide sequence composed of (A) and (B);

thereby generating a fourth pool of candidate nucleotide sequences;

(g) in the event that the length of the modified 5′

or modified 3′

candidate sequence is greater than 35 nucleotides, repeating step (e) one or more times wherein the modified 5′

candidate sequence or modified 3′

candidate sequence, respectively, has been trimmed by a greater number of nucleotides than in step (e) each time, until the length of the modified 5′

or modified 3′

candidate sequence is the earlier of (i) equal to, or (ii) lower than, 35 nucleotides;

(h) for each modified 5′

or modified 3′

candidate sequence of step (g) wherein;

(C) the modified 5′

or modified 3′

candidate sequence, and

(D) a 3′

or 5′

, respectively, candidate sequence or modified candidate sequence;

each have a melting temperature in the range of 78-83°

C. and both are derived from the same candidate nucleotide sequence,

adding to the third pool a modified candidate sequence composed of (C) and (D);

thereby generating a fifth pool of candidate nucleotide sequences; and

(i) optionally repeating steps (e)-(h) for one or more different candidate nucleotide sequences deleted in step (d),

thereby generating a sixth pool of candidate nucleotide sequences,

whereby the fourth, fifth and sixth pools consist of candidate nucleotide sequences composed of pairs of adjacent target-specific sequences for use in a probe pair hybridizable to the target mRNA.

8. The method of claim 7, which further comprises the step of outputting to a user interface device, a computer readable storage medium, or a local or remote computer system, or displaying, one or a plurality of candidate nucleotide sequences and/or modified candidate nucleotide sequences in the fourth, fifth and/or sixth pools and/or the 5′

candidate sequences or modified 5′

candidate sequences and/or 3′

candidate sequences or modified 3′

candidate sequences contained therein.

9. The method of claim 8, wherein the candidate nucleotide sequences and/or modified candidate nucleotide sequences are outputted as a pair of adjacent target-specific nucleotide sequences derived from said candidate nucleotide sequences and/or modified candidate nucleotide sequences, respectively.

10. The method of any one of claims 7 to 9, wherein a plurality of candidate nucleotide sequences and/or modified candidate nucleotide sequences in the fourth, fifth and/or sixth pools and/or the 5′

candidate sequences or modified 5′

candidate sequences and 3′

candidate sequences or modified 3′

candidate sequences contained therein are outputted or displayed in a ranked order based on a weighted score of the cross-hybridization potentials and the melting temperatures of said 5′

candidate sequences or modified 5′

candidate sequences and 3′

candidate sequences or modified 3′

candidate sequences.

11. The method of claim 10, wherein the weighted score is calculated according to the formula:

(Tm score*WFa)+(MCB score*WFb)+(PID score*WFc)

where;

Tm score is a melting temperature score calculated according to the formula;

(differential score+general score)/3

where the differential score is calculated according to the following formula;

1−

|(TmA−

TmB)|/5

where the general score is calculated according to the following formula;

(((80.5−

|(TmA−

80.5)|)/80.5)+(((80.5−

|(TmB−

80.5)|)/80.5)))

where TmA is the melting temperature of the 5′

candidate sequence or modified 5′

candidate sequence of a pair of adjacent target-specific sequences and TmB is the melting temperature of the 3′

candidate sequence or modified 3′

candidate sequence of said pair of adjacent target-specific sequences;

where;

MCB score is a maximum contiguous block score calculated according to the formula;

1−

(MCB/15);

where MCB is the greater of;

(i) the maximum contiguous block of identity between;

(A) a first target-specific nucleotide sequence in said pair of adjacent target-specific sequences; and

(B) a sequence in the database other than the complement of the target mRNA and, optionally, other than the complements of one or more alternatively spliced mRNAs corresponding to the same gene as the target mRNA;

and

(ii) the maximum contiguous block of identity between;

(A) a second target-specific nucleotide sequence in said pair of adjacent target-specific sequences; and

(B) a sequence in the database other than the complement of the target mRNA and, optionally, other than the complements of one or more alternatively spliced mRNAs corresponding to the same gene as the target mRNA;

where;

PID score is a percent identity score calculated according to the formula;

1−

(PID/85%);

where PID is the greater of;

(i) the greatest percentage sequence identity between;

(A) a first target-specific nucleotide sequence in said pair of adjacent target-specific sequences; and

(B) a sequence in the database other than the complement of the target mRNA and, optionally, other than the complements of one or more alternatively spliced mRNAs corresponding to the same gene as the target mRNA;

and

(ii) the greatest percentage sequence identity between;

(A) a second target-specific nucleotide sequence in said pair of adjacent target-specific sequences; and

(B) a sequence in the database other than the complement of the target mRNA and, optionally, other than the complements of one or more alternatively spliced mRNAs corresponding to the same gene as the target mRNA,

and wherein PIDco is the second predetermined cutoff,

and where WFa, WFb, and WFc are each independently a weighting factor, each of which is a real number.

13. A method for identifying a target-specific nucleotide sequence for use in a probe hybridizable to a target mRNA, comprising the steps of:

(a) generating a first pool of candidate nucleotide sequences of a first predetermined length or lengths that are reverse complements of a target mRNA sequence;

(b) deleting from said first pool one or more candidate nucleotide sequences that meet at least two of the following criteria;

(i) contains inverted repeats of greater than a predetermined length of consecutive nucleotides;

(ii) contains direct repeats of greater than a predetermined length of consecutive nucleotides;

(iii) has a GC content outside a predetermined range;

(iv) contains a contiguous stretch of C residues of greater than a predetermined length; and

(v) has a melting temperature that is outside a first predetermined melting temperature range;

thereby generating a second pool of candidate nucleotide sequences;

(c) deleting from said second pool one or more candidate nucleotide sequences that have a cross-hybridization potential to non-specific sequences that is higher than a predetermined threshold, thereby generating a third pool of candidate n sequences;

(d) deleting from said third pool one or more candidate nucleotide sequences that have a melting temperature outside a second predetermined temperature range, wherein the second predetermined melting temperature range is within the first predetermined melting temperature range;

(e) determining the melting temperature for a modified candidate nucleotide sequence, wherein the modified candidate nucleotide sequence is a modified form of a candidate nucleotide sequence deleted in step (d) because it has a melting temperature above the second predetermined range, wherein the modified candidate nucleotide sequence has been modified by trimming at least one nucleotide from the 5′

end or the 3′

end of said candidate nucleotide sequence;

(f) in the event that the modified candidate nucleotide sequence has a melting temperature within the second predetermined melting temperature range, adding to the third pool the modified candidate nucleotide sequence, thereby generating a fourth pool of candidate nucleotide sequences;

(g) in the event that the length of the modified candidate nucleotide sequence is greater than a second predetermined length, repeating step (e) one or more times wherein the modified candidate nucleotide sequence has been trimmed by a greater number of nucleotides than in step (e) each time, until the length of the modified candidate nucleotide sequence is the earlier of (i) equal to, or (ii) lower than, the second predetermined length;

(h) adding to the third pool each modified candidate nucleotide sequence of step (g) which has a melting temperature within the second predetermined melting temperature range;

thereby generating a fifth pool of candidate nucleotide sequences; and

(i) optionally repeating steps (e)-(h) for one or more different candidate nucleotide sequences deleted in step (d), thereby generating a sixth pool of candidate nucleotide sequences,

whereby the fourth, fifth and sixth pools consist of target-specific nucleotide sequences for use in a probe hybridizable to a target mRNA.

14. The method of claim 13, which further comprises the step of outputting to a user interface device, a computer readable storage medium, or a local or remote computer system, or displaying, one or a plurality of candidate nucleotide sequences and/or modified candidate nucleotide sequences in the fourth, fifth and/or sixth pools.

15. The method of any one of claims 13 and 14, wherein in step (c) a candidate target-specific sequence is deemed to have a cross-hybridization potential to non-specific sequences that is higher than said predetermined threshold if said candidate target-specific sequence has (i) a sequence percentage identity with a first sequence (hereinafter “

first non-target sequence”

) or its complement that is equal to or greater than a first predetermined cutoff, said first non-target sequence being other than the complement of the target mRNA and, optionally, other than the complements of one or more alternatively spliced mRNAs corresponding to the same gene as the target mRNA, and said first non-target sequence being present in a database comprising cellular mRNA sequences or cDNA sequences derived therefrom; and

(ii) a contiguous block of sequence identity with a second sequence (hereinafter “

second non-target sequence”

) or its complement that is equal to or greater than a second predetermined cutoff, said second non-target sequence being other than the complement of the target mRNA and, optionally, other than the complements of one or more alternatively spliced mRNAs corresponding to the same gene as the target mRNA, and said second non-target sequence being present in the database.

16. The method of any one of claims 13 and 14, wherein a plurality of candidate nucleotide sequences and/or modified candidate nucleotide sequences in the fourth, fifth and/or sixth pools are outputted or displayed in a ranked order based on a weighted score of the cross-hybridization potentials and the melting temperatures of said candidate nucleotide sequences and/or modified candidate nucleotide sequences.

17. The method of claim 15, wherein a plurality of candidate nucleotide sequences and/or modified candidate nucleotide sequences in the fourth, fifth and/or sixth pools are outputted or displayed in a ranked order based on a weighted score of the cross-hybridization potentials and the melting temperatures of said candidate nucleotide sequences and/or modified candidate nucleotide sequences.

21. The method of any one of claims 13 to 18, wherein in each step (g) of repeating the modified candidate nucleotide sequence is trimmed by one nucleotide.

27. The method of claim any one of claims 13 to 18, wherein the first predetermined length is selected from the range of 35 to 60 nucleotides.

28. The method of claim 27, wherein the second predetermined length is selected from the range of 30 to 45 nucleotides.

37. The method of any one of claims 4, 6, 15, and 17, wherein the first predetermined cutoff is selected from the range of 70-95% sequence identity.

38. The method of claim 37, wherein the first predetermined cutoff is selected from the range of 80-90% sequence identity.

39. The method of any one of claims 4, 6, 15, 17, 37 and 38, wherein the second predetermined cutoff is selected from the range of 10-18 contiguous nucleotides.

40. The method of claim 39, wherein the second predetermined cutoff is selected from the range of 14-16 contiguous nucleotides.

43. The method of any one of claims 4, 7, and 15, wherein the first non-target sequence and the second non-target sequence are the same.

52. A computer system for identifying a target-specific sequence for use in a probe hybridizable to a target mRNA, comprising:

a processor and a memory coupled with the processor, the memory storing a plurality of machine instructions that cause the processor to perform the method of any one of claims 13 to 17.