Tm-ENHANCED BLOCKING OLIGONUCLEOTIDES AND BAITS FOR IMPROVED TARGET ENRICHMENT AND REDUCED OFF-TARGET SELECTION

Abstract

The invention is directed to modified oligonucleotide compositions and methods for selectively reducing unwanted nucleic acid contaminants and enriching for desired nucleic acid targets from complex genomic nucleic acid mixtures for sequencing applications. The modified oligonucleotide compositions include one or more modified groups that increase the T m of the resultant oligonucleotide composition.

1 . An oligonucleotide for use in a selection method of a desired template nucleic acid, comprising an oligonucleotide having at least one T m -enhancing group,
 wherein the selection method comprises the hybrid capture method;   wherein the desired template nucleic acid comprises at least one member selected from a population of templates; and   wherein oligonucleotide is substantially complementary to at least one sequence of the desired template.   
     
     
         2 . (canceled) 
     
     
         3 . (canceled) 
     
     
         4 . (canceled) 
     
     
         5 . The oligonucleotide of  claim 1 , wherein the oligonucleotide comprises at least one member selected from a blocker or a bait. 
     
     
         6 . The oligonucleotide of  claim 1 , wherein the at least one T m -enhancing group is at least one member selected from the group consisting of a locked nucleic acid group, a bicyclic nucleic acid group, a C5-modified pyrimidine, a peptide nucleic acid group and combinations thereof. 
     
     
         7 . The oligonucleotide of  claim 1 , wherein the at least one T m -enhancing group comprises of a locked nucleic acid group, a bicyclic nucleic acid group or a combination thereof. 
     
     
         8 . The oligonucleotide of  claim 1 , wherein the at least one T m -enhancing group comprises a locked nucleic acid group or a bicyclic nucleic acid group. 
     
     
         9 . The oligonucleotide of  claim 8 , wherein the locked nucleic acid group or the bicyclic nucleic acid group comprises a nucleobase selected from the group consisting of cytosine, adenine and thymine. 
     
     
         10 . The oligonucleotide of  claim 1 , wherein the at least one T m -enhancing group provides an optimal enhanced T m  value in the range comprising from about 1.4° C. to about 25° C. 
     
     
         11 . (canceled) 
     
     
         12 . The oligonucleotide of  claim 1 , wherein the oligonucleotide comprises a blocker. 
     
     
         13 . The oligonucleotide of  claim 12 , wherein the blocker has substantial sequence complementarity to at least one sequence at a terminus of the desired template nucleic acid. 
     
     
         14 . The oligonucleotide of  claim 12 , wherein the blocker further comprises a barcode domain having a plurality of nucleotides. 
     
     
         15 . The oligonucleotide of  claim 14 , wherein the plurality of nucleotides comprises from about 5 to about 12 nucleotides arranged substantially contiguous. 
     
     
         16 . The oligonucleotide of  claim 14 , wherein the barcode domain comprises nucleotides having as nucleobases at least one member selected from the group selected from adenine, thymine, cytosine, guanine, inosine, 3-nitropyrrole, 5-nitroindole, and combinations thereof. 
     
     
         17 . The oligonucleotide of  claim 1 , wherein the oligonucleotide provides an improvement in the selection method of a desired template nucleic acid. 
     
     
         18 . The oligonucleotide of  claim 17 , wherein the improvement consists of an improved enrichment of the desire template nucleic acid relative to undesired template nucleic acids. 
     
     
         19 . The oligonucleotide of  claim 18 , wherein the improved enrichment comprises of an enrichment of at least 65%. 
     
     
         20 . The oligonucleotide of  claim 1 , further comprising a 3′-terminal modification. 
     
     
         21 . The oligonucleotide of  claim 20 , wherein the 3′-terminal modification prevents polymerase directed synthesis from the oligonucleotide. 
     
     
         22 . The oligonucleotide of  claim 20 , wherein the 3′-terminal modification comprises a 3′-deoxynucleotide, a 2′,3′-dideoxynucleotide or a 3′-spacer C3 group. 
     
     
         23 . The oligonucleotide of  claim 16 , further comprising a 3′-terminal modification. 
     
     
         24 . The oligonucleotide of  claim 23 , the 3′-terminal modification comprises a 3′-deoxynucleotide, a 2′,3′-dideoxynucleotide or a 3′-spacer C3 group. 
     
     
         25 - 80 . (canceled)