Microarray probe Tm matching by selective destabilization

Abstract

Methods and systems for designing oligonucleotide probes for use in microarray applications are provided herein. The described methods use duplex melting temperature (Tm) matching to destabilize the hybridization oligonucleotide probes to non-target sequences as compared to a target nucleotide sequence. Nucleic acid arrays containing probes selected by the described methods are also described.

1 . A method for designing a target-specific oligonucleotide probe comprising: 
 a) identifying a target-specific oligonucleotide probe comprising a sequence complementary to a target nucleotide sequence of interest and that has a computed T m  of about 65° C. or greater; and    b) modifying the sequence of the identified target-specific oligonucleotide probe to decrease the computed T m  so that the modified target-specific oligonucleotide probe hybridizes to at least one non-target nucleotide sequence with a computed T m  lower than the computed T m  of the hybridization of the modified target-specific oligonucleotide probe to the target nucleotide sequence.    
   
   
       2 . The method of  claim 1 , wherein the target-specific oligonucleotide probe hybridizes to the target nucleotide sequence at a computed T m  at about 75° C. or greater.  
   
   
       3 . The method of  claim 1 , wherein the target-specific oligonucleotide probe can hybridize to the target nucleotide sequence and one or more non-target nucleotide sequences.  
   
   
       4 . The method of  claim 1 , wherein designing the target-specific oligonucleotide probe further comprises: 
 a) identifying the at least one non-target nucleotide sequence that is homologous to the complement of the probe sequence; and    b) deleting or substituting at least one nucleotide in a region of the target specific oligonucleotide probe that is complementary to a region of the non-target nucleotide sequence that has the highest homology to the target nucleotide sequence to form a first modified target-specific oligonucleotide probe having a computed T m  to the at least one non-target nucleotide sequence that is the same or lower than the computed T m  of first modified target-specific oligonucleotide probe to the target nucleotide sequence.    
   
   
       5 . The method of  claim 4 , further comprising: 
 c) identifying a second non-target nucleotide sequence that has the second most homology to the target nucleotide sequence; and    d) modifying the first modified target-specific oligonucleotide probe by substituting or deleting at least one nucleotide in the sequence that is complementary to a region of the second non-target nucleotide sequence that has the second most homology to target nucleotide sequence to form a second modified probe having a computed T m  to the second non-target nucleotide sequence that is the same or lower than the computed T m  of the second modified probe to the target nucleotide sequence.    
   
   
       6 . A method of  claim 4 , further comprising: 
 a) identifying at least one homologous nucleotide sequence to the first modified target-specific oligonucleotide probe and identifying a second non-target nucleotide sequence that has the most homology to the complement of the first modified target-specific oligonucleotide probe; and    b) modifying the first modified target-specific oligonucleotide probe by substituting or deleting at least one nucleotide in the sequence of the first modified probe that is complementary to a region of the second non-target sequence that has the most homology to the complement of the first modified target specific oligonucleotide probe to form a second modified probe having a computed T m  to the second non-target nucleotide sequence the same or lower than that of the computed T m  of the second modified probe to the target nucleotide sequence.    
   
   
       7 . The method of  claim 1 , wherein the target-specific oligonucleotide probe is complementary to the target nucleotide sequence and at least two other non-target nucleotide sequences.  
   
   
       8 . The method of  claim 1 , wherein at least one nucleotide is deleted.  
   
   
       9 . The method of  claim 1 , wherein at least one nucleotide is substituted.  
   
   
       10 . The method of  claim 1 , wherein the target-specific oligonucleotide probe is at least 25 nucleotides long.  
   
   
       11 . The method of  claim 7 , wherein the target nucleotide sequence and the at least two other non-target nucleotide sequences have at least 80% homology over at least 25 nucleotides.  
   
   
       12 . The method of  claim 1 , wherein steps a) and b) are repeated until a computed T m  of the modified target-specific oligonucleotide probe to at least one of the non-target sequences has a decrease in computed T m  of at least 1° C.  
   
   
       13 . The method of  claim 1 , wherein the region of the non-target sequence that has the most sequence homology to the target sequence has a % GC content of at least 40%.  
   
   
       14 . The method of  claim 13 , wherein at least one nucleotide is substituted to in the target-specific oligonucleotide probe to eliminate a GC pair.  
   
   
       15 . The method of  claim 1 , wherein at least one nucleotide that is modified is located in the middle of the region of target-specific oligonucleotide probe that is complementary to the region of most homology between the target and non-target sequence.