Methods And Computer Systems For Identifying Target-Specific Sequences For Use In Nanoreporters

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.

What is claimed is: 
     
         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 . 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. 
     
     
         3 . 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.