Nucleic acid adaptors and uses thereof

Abstract

Provided herein are compositions, systems, methods, and kits for joining together the ends of one or more polynucleotides using at least one pair of blocking oligonucleotide adaptors. Blocking oligonucleotide adaptors can be used to reduce the formation of adaptor dimers or trimers (or higher-order concatemers) which can improve the yield of desirable polynucleotide-adaptor products in any recombinant nucleic acid workflow. Blocking oligonucleotide adaptors can comprise a double-stranded oligonucleotide adaptor (duplex) having an overhang cohesive portion that anneals with a blocking oligonucleotide which can be a separate single-stranded oligonucleotide. A blocking oligonucleotide, when annealed to an overhang portion, can prevent undesirable hybridization of the overhang portion to another nucleic acid, such as the overhang portion from another blocking oligonucleotide adaptor or a polynucleotide of interest.

What is claimed: 
     
         1 . A composition comprising:
 (1) a polynucleotide, wherein the polynucleotide comprises:
 a) a first double-stranded nucleic acid adaptor which is a first duplex having a first target-joining end and a first overhang end, wherein the first overhang end contains a first overhang nucleic acid sequence; 
 b) a second double-stranded nucleic acid adaptor which is a second duplex having a second target-joining end and a second overhang, wherein the second overhang end contains a second overhang nucleic acid sequence, wherein the first and second overhang nucleic acid sequences are at least partially complementary to each other; and 
 c) a linear double-stranded target polynucleotide having a first end and a second end, wherein the first end is joined to the first target-joining end of the first double-stranded nucleic acid adaptor, and the second end is joined to the first target-joining end of the second double-stranded nucleic acid adaptor 
   and   (2) a first blocking oligonucleotide that is capable of annealing to at least a portion of the first overhang nucleic acid sequence, and a second blocking oligonucleotide that is capable of annealing to at least a portion of the second overhang nucleic acid sequence;
 wherein:
 (i) the first overhang nucleic acid sequence is or is not annealed to the first blocking oligonucleotide which, when annealed forms a third duplex, 
 (ii) the second overhang nucleic acid sequence is or is not annealed to the second blocking oligonucleotide which, when annealed forms a fourth duplex; and 
 (iii) the melting point of the first duplex is higher than the melting point of the third duplex and the melting point of the second duplex is higher than the melting point of the fourth duplex. 
 
   
     
     
         2 . The composition of  claim 1 , wherein the first and second overhang ends of the double-stranded nucleic acid adaptors of the polynucleotide are 5′ overhang ends or 3′ overhangs. 
     
     
         3 . The composition of  claim 1 , wherein the first overhang nucleic acid sequence and the second overhang nucleic acid sequence of the double-stranded nucleic acid adaptors of the polynucleotide are each about 1-20 nucleotides in length. 
     
     
         4 . The composition of  claim 1 , wherein the strands of the first duplex and of the second duplex that comprise the first and second overhang nucleic acid sequences, respectively, of the polynucleotide are each about 2-25 nucleotides in length. 
     
     
         5 . The composition of  claim 1 , wherein the first blocking oligonucleotide and the second blocking oligonucleotide are each the same length or shorter than the overhang nucleic acid sequence to which it is capable of annealing. 
     
     
         6 . The composition of  claim 1 , wherein the first overhang nucleic acid sequence is annealed to the first blocking oligonucleotide, the second overhang nucleic acid sequence is annealed to the second blocking oligonucleotide and the polynucleotide is a linear polynucleotide. 
     
     
         7 . The composition of  claim 1 , wherein the first overhang nucleic acid sequence is not annealed to the first blocking oligonucleotide, the second overhang nucleic acid sequence is not annealed to the second blocking oligonucleotide, and the overhang end of the first double-stranded nucleic acid adaptor is hybridized to the second overhang end of the second double-stranded nucleic acid adaptor to form a circular double-stranded polynucleotide having two nicks, wherein the nicks are on opposite strands and at different positions of the polynucleotide. 
     
     
         8 . The composition of  claim 7 , wherein a nucleotide at the position of each nick lacks a 5′ phosphate group. 
     
     
         9 . The composition of  claim 7 , further comprising a nick translation reaction mixture. 
     
     
         10 . The composition of  claim 9 , wherein the nick translation reaction mixture comprises (i) a coupled 5′ to 3′ DNA polymerization/degradation reaction mixture, or (ii) a coupled 5′ to 3′ DNA polymerization/strand displacement reaction mixture. 
     
     
         11 . The composition of  claim 9 , wherein the nick translation reaction mixture comprises a DNA polymerase enzyme. 
     
     
         12 . The composition of  claim 9 , wherein the nick translation reaction mixture comprises an enzyme selected from the group consisting of  E. coli  DNA polymerase I, Taq DNA polymerase, Vent DNA polymerase, Klenow DNA polymerase I, Tfi DNA polymerase, Bst DNA polymerase, and phi29 DNA polymerase. 
     
     
         13 . The composition of  claim 9 , wherein the nick translation reaction mixture comprises deoxyribonucleoside triphosphates. 
     
     
         14 . The composition of  claim 1 , wherein the first double-stranded nucleic acid adaptor of the polynucleotide comprises biotin, or the second double-stranded nucleic acid adaptor comprises biotin, or both the first and second double-stranded nucleic acid adaptors comprise biotin. 
     
     
         15 . The composition of  claim 7 , further comprising one or more endonucleases and/or exonucleases. 
     
     
         16 . The composition of  claim 15 , comprising a T7 exonuclease. 
     
     
         17 . The composition of  claim 15 , comprising an S1 nuclease. 
     
     
         18 . The composition of  claim 1 , wherein the first duplex comprises a sequence of SEQ ID NO:1 annealed to a sequence of SEQ ID NO: 2 and the first blocking oligonucleotide comprises a sequence of SEQ ID NO: 3. 
     
     
         19 . The composition of  claim 18 , wherein the second duplex comprises a sequence of SEQ ID NO:6 annealed to a sequence of SEQ ID NO: 25 and the second blocking oligonucleotide comprises a sequence of SEQ ID NO: 4.