Methods and computer systems for identifying target-specific sequences for use in nanoreporters
View Patent ↗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.
1. A method comprising the steps of:
(a) generating candidate target-specific nucleotide sequences that are reverse complements of a target mRNA sequence;
(b) dividing each target-specific nucleotide sequence into two nucleotide sequences of equal length consisting of a 5′ sequence and a 3′ sequence, thereby generating a first pool of adjacent target-specific sequence pairs;
(c) deleting from said first pool one or more adjacent target-specific sequence pairs if either sequence of the sequence pair meets any of the following criteria: (i) contains inverted repeats of greater than 7 consecutive nucleotides; (ii) contains direct repeats of greater than 9 consecutive nucleotides; (iii) contains a GC content outside a range of 30-70%; (iv) contains contiguous stretches of C residues of greater than 3 nucleotides in length; and (v) has a melting temperatures outside a first melting temperature range of 70-90° C.; thereby generating a second pool of adjacent target-specific sequence pairs;
(d) deleting from said second pool one or more adjacent target-specific sequence pairs if either sequence of the sequence pair has a cross-hybridization potential to non-specific sequences that is 85% or greater, thereby generating a third pool of adjacent target-specific sequence pairs;
(e) deleting from said third pool one or more adjacent target-specific sequence pairs if either sequence of the sequence pair has a melting temperature outside a second melting temperature range of 78-83° C., thereby generating a fourth pool of adjacent target-specific sequence pairs;
(f) generating a fifth pool of adjacent target-specific sequence pairs, said fifth pool comprising the adjacent target-specific sequence pairs deleted from said third pool;
(g) determining the melting temperature of each sequence of the adjacent target-specific sequence pairs of said fifth pool;
wherein if both sequences in an adjacent target-specific probe pair have a melting temperature below the second melting temperature range of 78-83° C., said adjacent target-specific probe pair is deleted from the fifth pool;
wherein if one or both sequences in an adjacent target-specific probe pair have a melting temperature above the second melting temperature range of 78-83° C., one or both sequences are trimmed until one or both of said sequences are within the second melting temperature, thereby generating a sixth pool of adjacent target-specific sequence pairs;
wherein if one sequence in an adjacent target-specific probe pair has a melting temperature below the second melting temperature range of 78-83° C., and the other sequence in said pair is within or above the second melting temperature, said sequence with the low melting temperature is extended until said sequence is within the second melting temperature, thereby generating a seventh pool of adjacent target-specific sequence pairs;
(h) selecting and producing one or more adjacent target-specific sequence pairs from said fourth, sixth and seventh pools for use as a probe pair hybridizable to a target mRNA, thereby producing adjacent target-specific probe pairs;
(i) hybridizing at least one probe of each said one or more adjacent target-specific probe pairs, selected and produced in step (h), to at least a first label attachment region, comprising a DNA sequence having a regularly repeated base every about 4 to about 25 bases, and comprising a RNA molecule having a regularly repeated base every about 4 to about 25 bases which is hybridized to the DNA sequence, to which RNA molecule are attached at said regularly repeated base one or more label monomers that emit light constituting at least a first signal; and,
(j) contacting an adjacent target specific probe pair from step (i) with a target mRNA under conditions sufficient to permit hybridization of said adjacent target specific probe pair and said 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 more of the adjacent target-specific sequence pairs in said fourth pool.
3. The method of claim 1 , wherein steps (a) through (j) are repeated to produce a plurality of adjacent target-specific probe pairs, wherein each adjacent target-specific probe pair is hybridizable to a different target mRNA sequence.
4. The method of claim 1 , wherein the length of the candidate target-specific nucleotide sequences is selected from the range of 70 to 120 nucleotides.
5. The method of claim 1 , wherein the predetermined range of GC content of step (c)(iii) is 40-70%.
6. The method of claim 1 , wherein the target mRNA is an alternatively spliced mRNA.
7. The method of claim 1 , wherein said 5′ sequence of adjacent target-specific sequence pair is trimmed from the 5′ end.
8. The method of claim 1 , wherein said 3′ sequence of adjacent target-specific sequence pair is trimmed from the 3′ end.
9. The method of claim 1 , wherein said 5′ sequence of adjacent target-specific sequence pair is extended from the 5′ end.
10. The method of claim 1 , wherein said of the 3′ sequence of adjacent target-specific sequence pair is extended from the 3′ end.